Function And Mechanisms Of ER-localized Protein FKBP9 In Glioma

Background:Endoplasmic reticulum(ER)is an important organelle for the synthesis of proteins,lipids and carbohydrates in eukaryotic cells.Multiple internal and eternal stimuli on tumor cells,such as oxidative stress,nutritional deficiency,and ultraviolet irradiation,result in the accumulation of large amounts of unfolded or misfolded proteins in ER,i.e.,ER stress.On the one hand,the unfolded protein response(UPR)induced by ER stress can limit the synthesis of new proteins,extend the protein-folding capacity of ER,and accelerate the degradation of unfolded or misfolded proteins,thereby promoting tumor cells survival,growth and protective autophagy.On the other hand,prolonged unfolded protein response will lead to apoptosis or autophagic death of tumor cells.Therefore,ER stress plays a dual role in tumorigenesis and development,making it a research hotspot and an increasingly important target for tumor therapy.Gliomas are the most common primary malignant brain tumors,and there is a lack of effective treatment clinically due to its high heterogeneity,high infiltration and extremely high recurrence.Studies have found that the ER stress pathway is closely related to the protein homeostasis in glioma cells.Recently,many drugs and treatments targeting UPR have been investigated in clinical research.FKBP(FK506 binding protein)family is a class of proteins that can specifically bind to immunosuppressive agents,which are widely present in different tissues.FKBPs have peptidyl-prolyl cis-trans isomerase(PPIase)activity and generally contain three domains: TPR(tetratricopeptide repeat domain),FKBP?C(FK506 like binding protein c-terminal domain)and TMD(transmembrane domain).Studies have shown that FKBPs are involved in a variety of signaling pathways in tumor cell growth,survival and apoptosis,especially in tumor development and chemotherapy resistance.In glioma,FKBP12 as an m TOR interacting protein binds with rapalink-1 to inhibit glioma.In addition,FKBP52 is negatively correlated with TDO(tryptophan-2,3-dioxygenase)in glioma and in normal brain tissue,and the inhibition of TDO expression and activity by dexamethasone is dependent on FKBP52.The antiapoptotic protein FKBP14 plays a decisive role in the resistance to erlotinib of glioma.FKBP5 promotes glioma growth and drug resistance by activating NF-?B pathway.FKBP9 localizes in ER.Data showed that FKBP9 is highly expressed in a variety of cancers including glioma,pancreatic cancer,renal cancer,melanoma,suggesting that it may play an important role in related tumors.Notably,integrated genomic analysis showed that FKBP9,together with TP53,PTEN,EGFR,are amplificated and mutated in glioma,and such mutation might be related to the identification of different glioma subtypes.However,there are few reports on the role of FKBP9 in tumors including glioma,and the function and mechanisms of FKBP9 in glioma are unclear.Objective:The purpose of our study is to dissect the function and mechanisms of FKBP9 in glioma.By constructing glioma cells with stable knockdown or overexpression of FKBP9,we explore the relationship between ER-localized protein FKBP9 and ER stress,and the regulation mechanisms of FKBP9 itself.This study will provide a novel therapeutic avenue for the treatment of glioma.Methods:1.Clinical data analysis2.Protein expression in clinical tissue samples by immunohistochemical assay3.Plasmid construction of FKBP9 wild-type,deletion and mutantion4.Stable knockdown of FKBP9 by lentivirus infection,and transient expression of FKBP9 with adenovirus5.CCK8 cell viability assay,colony formation assay,Transwell invasion assay,and sphere formation in 3D culture system6.Chick embryo chorioallantoic membrane invasion model7.Subcutaneous tumor formation model in nude mice8.Protein interaction by immunoprecipitation9.Protein co-localization by immunofluorescence Results:1.Clinical significance of FKBP9 in glioma1)High expression of FKBP9 in glioma was associated with poor prognosis.2)FKBP9 expression was related to the degree of malignancy of glioma.The higher the clinical stage and grade corresponded to,the higher the expression of FKBP9.2.Detection of FKBP9 in glioma cells1)FKBP9 was highly expressed in most of the tested glioma cell lines and located in the intracellular ER.2)The protein expression level of FKBP9 in glioma under 3D culture conditions cells was significantly higher than that in 2D.3.Knockdown FKBP9 inhibits glioma in vitro and in vivo.1)Knockdown of FKBP9 inhibited glioma cells proliferation.2)Knockdown of FKBP9 inhibited glioma cells invasion.3)Knockdown of FKBP9 inhibited glioma cells sphere formation,i.e.,anchor independent growth.4)Knockdown of FKBP9 led to glioma cells more sensitive to TMZ.5)Knockdown of FKBP9 inhibited glioma cell invasion in vivo.6)Knockdown of FKBP9 inhibited glioma growth in vivo.4.The FKBP9-M541 I mutation displays a weaker carcinogenesis compared with the wild type.1)Overexpression of FKBP9 promoted glioma cells proliferation,while overexpression of FKBP9-M541 I has no such effect.2)Overexpression of FKBP9 promoted sphere formation of glioma cells,while overexpression of FKBP9-M541 I has no such effect.3)Overexpression of FKBP9 promoted tumorigenesis in vivo,while overexpression of FKBP9-M541 I has no such effect.5.FKBP9 activates p38 signaling pathway.Through Western Blot detection of key proteins in MAPK,AKT-m TOR,Wnt and other pathways,we found that overexpresssion of FKBP9 in glioma cells up-regulated p38 phosphorylation,while FKBP9 knockdown led to a decrease in p38,which was rescued by adenovirus-mediated overexpression of FKBP9.6.The connection between FKBP9 and ER stress.1)Loss of FKBP9 induced ER stress.Knockdown of FKBP9 increased the transcriptional activity of ATF6,augmented the transcriptional levels of DDIT3,DNAJB9 and SEL1 L,which are the ER stress downstream genes,and activated the IRE1? –XBP1 pathway.2)Overexpression of FKBP9 resisted cell death triggered by ER stress.Upon treatment with the ER stress inducer Tg(Thapsigargin),the anti-apoptotic proteins of Bcl-2 family significantly increased in FKBP9-WT overexpressed cells compared to control cells.7.FKBP9 interacts and co-localizes with Bi P.8.FKBP9 degradation via ubiquitination during ER stress.1)ER stress inducers BFA(Brefeldin A),Tg(Thapsigargin)and Tm(Tunicamycin) reduced the protein expression levles of FKBP9,while m RNA level of FKBP9 was not affected.2)Both the ER stress inhibitors and UPR pathway inhibitors pretreated the down-regulation of FKBP9 caused by the ER inducers.3)Tg-induced reduction of FKBP9 protein level was blocked by the proteasome inhibitor MG132.FKBP9 was ubiquitinated during ER stress with Lysine 265 as the key site of ubiquitination.In sum,Our study found that FKBP9 promoted glioma cells proliferation,stemness and tumor growth in vitro and in vivo.Knockdown of FKBP9 caused ER stress,while overexpression of FKBP9 resisted cell death induced by ER stress.And a change in the ubiquitination of FKBP9 was observed under this circumstance.Conclusion:1.FKBP9 promotes glioma proliferation,invasion,anchor independent growth,and tumorigenesis in vitro and in vivo,possibly by activating the p38 pathway.2.FKBP9 participates in the regulation of ER stress pathway by interacting with BiP.3.FKBP9 can be degraded via ubiquitination under ER stress.