The Study Of Mechanism Of Autophagy Inhibition By TOPK Phosphorylating ULK1 To Promote Glioma Resistance To TMZ

Backgroud: Glioma is the most malignant brain tumor.It is difficult to be completely resected and is easy to be resistant to radiotherapy and chemotherapy.Abnormal autophagy is one of the causes of glioma resistance to chemoradiotherapy,but its regulation mechanism is still unclear.Studies have shown that T-lymphocyte-derived protein kinase(TOPK)is specifically expressed in glioma stem cells,and is inseparable from glioma chemoradiation resistance and recurrence,but the mechanism is still unclear.Objective: 1.To define the relationship between TOPK and autophagy.2.To elucidate the mechanism by which TOPK regulates autophagy.3.To explore the role of TOPK regulating autophagy in the resistance of glioma to temozolomide(TMZ).Methods: 1.The glioma clinical specimens and human glioma cell lines were collected,and autophagy active marker molecules(LC3-II and P62)were detected by Western Blot.2.Silencing or overexpression method was used to reduce or increase TOPK expression.TOPK specific inhibitor was used to inhibit its activity.Western Blot was used to detect autophagy active marker molecules(LC3-II and P62).Transmission electron microscopy and fluorescence experiments were used to observe autophagosomes.3.Bioinformatics technology was used to predict the potential substrates of TOPK,including ULK1.4.The eukaryotic expression plasmids of three functional domains of ULK1 were constructed.The interaction and binding domain of TOPK and ULK1 were confirmed by Pull down assay.5.NetPhos 2.0 was used to predict potential phosphorylation sites of ULK1,and the corresponding peptides were synthesized in vitro.6.In vitro kinase assay and mass spectrometry assay were used to confirm that TOPK could directly phosphorylate ULK1 and to identify specific phosphorylation sites.7.In TOPK silencing,activity inhibition and overexpression cells,the stability of ULK1 and the phosphorylation levels of Beclin-1 and Atg13(endogenous substrates of ULK1)were detected by Western Blot.8.The sites of ULK1 phosphorylated by TOPK were mutated by site-directed mutagenesis.The inactivated mutant eukaryotic expression plasmid was constructed and transfected into 293 T cells.Western Blot was used to detect ULK1(representing its stability)and phosphorylation Beclin-1 and phosphorylation of Atg13(representing ULK1 activity),LC3-II(representing autophagy activity).9.TOPK,ubiquitin and wild-type or mutant ULK1 were co-transfected into 293 T cells,and the ubiquitination level of ULK1 was detected by immunoprecipitation.10.Combination of TMZ and TOPK inhibitors to treat TOPK high expressing glioma cells or TMZ for the treatment of TOPK silencing cell lines,Western Blot was used to detect the apoptotic molecule: Caspase-3;MTT assay and softagar colony assay were used to analyze cell proliferation and cell anchorageindependent growth.11.Stable expression of wild-type or mutant ULK1 in TOPK high expression cells,and and with TMZ treatment,Western Blot was used to detect the apoptotic molecule,Caspase-3;MTT assay and softagar colony assay were used to analyze cell proliferation and cell anchorage-independent growth.Results: 1.High-grade gliomas expressed high levels of TOPK and P62,and low level of LC3-II.2.The glioma cell lines with high expression of TOPK exhibited high level of P62 and low level of LC3-II.3.Inhibiting TOPK activity or silencing TOPK promoted LC3-II expression,and inhibited P62 expression,while overexpressing TOPK inhibited LC3-II expression,and promoted P62 expression.4.Silencing TOPK expression increased the number of autophagosomes.5.As a new substrate of TOPK,ULK1 interacted with TOPK by its SPR domain.6.TOPK phosphorylated ULK1 at Ser469,Ser495 and Ser533 in vitro.7.Mass spectrometry assay confirmed that the S469 phosphorylation of ULK1 was present in the cells.8.Inhibition of TOPK enhanced ULK1 activity and stability,while overexpression of TOPK attenuated ULK1 activity and stability.9.Inactivation mutation of Ser469,Ser495 and Ser533 of ULK1 increased its activity,prolonged its half-life,and up-regulated the expression of LC3-II.10.Glioma cells were treatd with the combination of TMZ and TOPK inhibitors promoted apoptosis,inhibited cell proliferation and colony formation;TOPK silencing cell lines were treated with TMZ obtained the same results.11.Cell lines stably expressing Ser469,Ser495 and Ser533 inactivating mutant ULK1 treated with TMZ increased apoptosis,and decreased cell proliferation and anchorage-independent growth.Conclusion: 1.TOPK inhibited autophagy.2.TOPK interacted with the SPR domain of ULK1 and phosphorylateed ULK1 at Ser469,Ser495 and Ser533,reducing its activity and stability,thereby inhibiting the initiation of autophagy.3.Phosphorylation of ULK1 by TOPK inhibited autophagy activity and promoted glioma resistance to TMZ.