The Mechanism Of NUSAP1 In Regulating Glioblastoma Growth And Proliferation By Stabilizing ATR

Glioblastoma(Glioblastoma multiforme,GBM),also called grade IV astrocytoma,along with other gliomas,constitutes the vast majority of malignant brain tumors.GBM is the most aggressive brain tumor with high mortality,poor prognosis and short survival.It is urgent to explore more therapeutic options for GBM diagnosis,therapy and prognosis to further prolong the survival of GBM patients.NUSAP1 is firstly found as a mitotic regulator,NUSAP1 is also a microtubule-associated protein.NUSAP1 involves not only in mitosis,but also plays essential roles in diverse biological processes,especially in cancer biology.NUSAP1 elevates in various human tumors.NUSAP1 thereby acts as a potential target in cancer therapy.However,the mechanism of this gene in GBM has not been well described.This study aims to determine the role of NUSAP1 in the tumorigenesis of GBM.R2 Genomics Analysis and clinical samples analysis reveal that NUSAP1 acts as a biomarker for diagnosis and therapy in GBM.According to the investigation on in vitro and in vivo,NUSAP1 promotes GBM growth.Besides,NUSAP1 potentiates chemoresistance in GBM,which provide the foundation of NUSAP1 in GBM therapy.The main results are listed as below:1.NUSAP1 is highly expressed in glioma and its highly expression correlates with poor prognosis of glioma patientsTo investigate the role of NUSAP1 in GBM,an immunochemistry(IHC)assay was performed to detect the expression of NUSAP1 in 8 normal brain tissues and 64 glioma samples.NUSAP1 was highly expressed in glioma samples in a grade-dependent manner.The level of NUSAP1 in 4 GBM cell lines including A172,U-118 MG,U-87 MG and LN-229 was detected.The results showed that NUSAP1 was highly expressed in all of these cell lines.We further examined the expression of NUSAP1 in a pair of peritumoral and tumor tissues,and NUSAP1 was upregulated in the tumor tissue.To further determine the role of NUSAP1 in the prognosis of GBM patients,the prognostic value of NUSAP1 was analyzed in the R2: Genomics Analysis and Visualization Platform.In comparison with a normal dataset,the level of NUSAP1 was upregulated in 12 glioma datasets.According to 3 different datasets,the expression of NUSAP1 was highly expressed in dead patients compared with alive patients.In addition,a high level of NUSAP1 predicted poor prognosis in glioma patients.In consistent with the results of IHC,NUSAP1 expressed in glioma patients in a grade-dependent manner.These results indicate that NUSAP1 plays important role in GBM.NUSAP1 is regarded as a molecular target in GBM diagnosis and therapy.2.NUSAP1 regulates cell proliferation and apoptosis in GBM cellsA high level of NUSAP1 in GBM indicates that NUSAP1 acts as an indicator for the diagnosis prognosis and therapy of GBM.These results suggested that NUSAP1 serves as a tumor promoter in GBM.To confirm this hypothesis,three independent short hairpin RNAs(sh RNAs)were designed to knock down NUSAP1 in the GBM cell lines U-87 MG,LN-229 and A172.Western blot analysis showed that NUSAP1 was successfully knocked down by these 3 sh RNAs.MTT assays were performed,results showed that these 3 sh RNAs inhibits cell proliferation in GBM cells.Since sh NUSAP1#1 presented the highest efficiency,sh NUSAP1#1 was then used to downregulate the expression of NUSAP1 in subsequent experiments.Brd U assays indicate that deletion of NUSAP1 inhibits the ability of DNA synthesis in GBM cells.By microscopy,GBM cells with NUSAP1 knockdown showed significant morphological changes,and the cell numbers sharply decreased.After knocking down NUSAP1 in GBM cell lines,cells died significantly.We therefore examined apoptosis by flow cytometry.The proportion of apoptotic cells increased in cells with NUSAP1 knockdown.To further validate the results above,Western blotting was performed.The levels of the apoptosis-related protein bcl2 and the apoptotic marker cleaved caspase-3 were distinctly altered,with bcl2 decreasing and cleaved caspase-3 increasing upon NUSAP1 depletion.Additionally,caspase-3/7 activity was detected in U-87 MG,LN-229 and A172 cells.Consistent with the results obtained above,caspase-3/7 activity increased sharply after knocking down NUSAP1.These results remind us that depletion of NUSAP1 inhibits cell proliferation and induces apoptosis in GBM cells.3.NUSAP1 regulates DNA damage in GBM cellsWe constructed a mini ontology in French's database using gene sets related to NUSAP1,which were identified by a significant difference of P<0.001.We found that NUSAP1 correlated with the DNA repair process significantly.We hypothesized that NUSAP1 acted as a regulator in the DDR to dictate cell fate.To confirm this hypothesis,a comet assay was performed to assess DNA damage through single-cell gel electrophoresis.A large number of cells with tailed DNA were observed after knocking down NUSAP1.Phosphorylated histone H2AX(?-H2AX)is an indicator of the DDR.?-H2 AX is recruited to lesions when DNA damage occurs.An immunofluorescence(IF)assay was performed to examine ?-H2 AX levels.Positive cell staining for ?-H2 AX increased in cells with NUSAP1 knockdown.To further validate the results acquired above,the active forms of proteins involved in the DDR were detected,and the results showed that P-Chk1,P-Chk2 and ?-H2 AX levels were upregulated in cells with NUSAP1 knockdown.4.NUSAP1 regulates colony formation in vitro and tumorigenesis in vivo in GBM cells.To further validate whether NUSAP1 affects clonogenicity in GBM cells,a soft agar assay was performed,and the results demonstrated that downregulation of NUSAP1 inhibited the ability of colony formation.In addition,the effects of NUSAP1 on tumorigenesis were measured in an orthotopic implantation model.The results showed that NUSAP1 depletion significantly inhibited tumor formation in vivo.Furthermore,NUSAP1 depletion clearly prolonged the survival of the mice.5.NUSAP1 regulates GBM growth by stabilizing ATR.During the detection of DDR-related proteins,we found that ATR levels decreased significantly in cells with NUSAP1 knockdown.To further investigate whether NUSAP1 regulates GBM growth by ATR,NUSAP1 was stably overexpressed in the U-87 MG,LN-229 and A172 cell lines.ATR levels increased after NUSAP1 overexpression.Interestingly,knocking down NUSAP1 did not affect the m RNA level of ATR.NUSAP1 overexpression did not affect the m RNA level of ATR as well.We assume that NUSAP1 regulates ATR through posttranslational modification(PTM).The effects of NUSAP1 depletion on ATR levels were measured by CHX chase assay.As expected,knocking down NUSAP1 significantly decreased the stability of ATR.In contrast,NUSAP1 overexpression stabilizes ATR.Moreover,we performed a ubiquitin assay to detect the ubiquitination level of ATR.The results indicated that the ubiquitination level of ATR increased after knocking down NUSAP1,while the ubiquitination level of ATR decreased when NUSAP1 overexpressed.These results indicate that NUSAP1 reduces the ubiquitination level of ATR to stabilize ATR.To investigate whether NUSAP1 plays the roles mentioned above by stabilizing ATR,ATR was overexpressed after knocking down NUSAP1,and the results showed that ATR rescued the expression of ?-H2 AX and cleaved-caspase9 in cells with NUSAP1 knockdown.These results remind us that NUSAP1 is involved in GBM growth by stabilizing ATR.6.NUSAP1 stabilizes ATR through its SAP domain to promote sumoylation of ATR.To further investigate the role of NUSAP1 in stabilizing the ATR protein,we detected the interaction of NUSAP1 and ATR.immunoprecipitation(IP)assay was performed.As expected,both endogenous and exogenous NUSAP1 interacted with ATR.Furthermore,truncated forms of NUSAP1 were co-transfected with full-length ATR.The results indicated that the COOH-terminal of NUSAP1 interacted with ATR.Previous studies have demonstrated that NUSAP1 contains an SAP domain.SAP domains are commonly found in many SUMO E3 ligases.SAP domains have also been implicated in substrate recognition and ligase activity.Sumoylation has been indicated to antagonize the ubiquitin-dependent degradation.To detect whether the SAP domain of NUSAP1 regulates sumoylation to stabilize ATR,NUSAP1-?SAP with deletion of its SAP domain was constructed.Results indicate that NUSAP1-?SAP overexpression decreased ATR levels compared with the NUSAP1 overexpression group.Furthermore,NUSAP1-?SAP overexpression reversed the sumoylation and ubiquitination of ATR induced by NUSAP1 overexpression.These results indicate that NUSAP1 stabilizes ATR through its SAP domain.7.NUSAP1 regulates chemotherapeutic resistance through its SAP domain.NUSAP1 regulates the stability of ATR in GBM cells according to the results mentioned above.ATR acts as an essential factor in DNA damage as well as the DDR and plays important roles in chemotherapeutic resistance.To validate whether NUSAP1 is involved in the regulation of chemotherapeutic sensitivity,we treated GBM cells with 2 chemotherapeutic agents,TMZ and DOX.After knocking down NUSAP1,cells treated with both TMZ and DOX showed higher apoptosis and lower cell viability.NUSAP1 depletion resulted in a lower IC50 towards both TMZ and DOX than the control group.In contrast,cells with NUSAP1 overexpression had lower apoptosis and higher cell viability after treatment with these 2 agents.Deletion of the SAP domain restored these effects.NUSAP1 overexpression showed a higher IC50 towards TMZ and DOX,while deletion of the SAP domain showed a lower IC50 than the NUSAP1-overexpressing group.These results suggested that NUSAP1 regulates chemoresistance of GBM cells,and its SAP domain plays important role in this process.Overall,NUSAP1 is highly expressed in GBM tissues in a grade-dependent manner compared with normal brain tissues.NUSAP1 is also highly expressed in glioma patients,dead patients and in glioma cells.In addition,NUSAP1 participates in cell proliferation,apoptosis as well as in DNA damage of GBM cells.In terms of mechanism,SAP domain of NUSAP1 promotes the sumoylation of ATR and thereby stabilizes ATR.Significantly,NUSAP1 potentiates chemotherapeutic resistance of temozolomide(TMZ)and doxorubicin(DOX)through its SAP domain.These results indicate that NUSAP1 promotes GBM cell growth,which provides feasible basis for NUSAP1 in GBM therapy.