Role Of Hsp90 Chaperone In Glioma Harboring FGFR3-TACC3 Fusion Gene

Objective: The study was designed to gain more insight on previously reported genomic event that frequently occurs in glioblastoma,the gene fusion of FGFR3 and TACC3(FGFR3-TACC3).A previous study has shown an epigenetic regulation of mi R-99 a on the FGFR3-TACC3 fusion gene.This study is focused on the post-translational regulatory mechanisms of the FGFR3-TACC3 fusion protein,identifying the potential therapeutic targets for the FGFR3-TACC3 fusion gene,providing new clues for clinical treatment.First,by analyzing the proteomic data of FGFR3-TACC3,proteins that strongly interact with the FGFR3-TACC3 fusion protein were identified and the key post-transcriptional regulatory mechanisms were suggested.Then,exploring the role of this post-transcriptional regulation in the initiation and development of FGFR3-TACC3 fusion gene positive glioblastoma.Finally,to find new therapeutic targets and new treatment strategies for the FGFR3-TACC3 fusion gene based on the findings of the proteomic study.Methods: To explore the potential post-translational regulatory mechanism of the FGFR3-TACC3 fusion protein,recombinant expression plasmids of pc DNA3.1-HA-FGFR3-TACC3 and pc DNA3.1-HA-FGFR3 were transiently transfected into U-251 MG glioblastoma cells and stably transfected cells expressing FGFR3-TACC3 and FGFR3 were selected using G418.Stably transfected cell lysates were extracted.HA-tagged FGFR3-TACC3 and FGFR3 proteins were immuno-precipitated and analyzed by two-dimensional liquid chromatography-tandem mass spectrometry to reveal potential binding partners.Reverse immunoprecipitation was applied to verify the proteomics results to determine the proteins that strongly interact with the FGFR3-TACC3 fusion protein.Key post-translational regulatory mechanisms were suggested based on proteomics results.Proteomics studies confirmed that the FGFR3-TACC3 fusion protein formed a ternary complex with chaperone Hsp90 and co-chaperone Cdc37,suggesting that the Hsp90 chaperone system is involved in the post-translational regulation of the FGFR3-TACC3 fusion protein.To further investigate the role of the Hsp90 chaperone system in the post-translational regulation of the FGFR3-TACC3 fusion protein,the Hsp90 inhibitors Onalespib,17-AAG,and si RNA of Cdc37 were applied.FGFR3-TACC3 protein expression level and phosphorylation level(Y647,Y648)were detected by immunoblot.Under the treatment of the Hsp90 inhibitor,the membrane expression level of FGFR3-TACC3 was measured by flow cytometry and membrane protein extraction kit,respectively.To explore the post-translational regulatory mechanism of the Hsp90 molecular chaperone system in glioblastoma harboring the FGFR3-TACC3 fusion gene,immunoblot and co-immunoprecipitation techniques were used to detect the ubiquitination of FGFR3-TACC3 protein upon Hsp90 inhibition.By using the protein synthesis inhibitor,the half-life of FGFR3-TACC3 protein was observed with or without Hsp90 inhibitors.To investigate the role of the Hsp90 molecular chaperone system in the initiation and development of FGFR3-TACC3 fusion gene positive glioblastoma,we first set up gradient drug concentrations and measured the proliferation of FGFR3-TACC3 tumor cells under a certain drug concentration using the cell proliferation-toxicity test kit,then the half inhibitory concentration of Hsp90 inhibitor was determined.Besides,clonogenic assay was used to determine the ability of FGFR3-TACC3 tumor cells to form resistance colonies under Hsp90 inhibitors drug treatment and colony formation rate was calculated.To explore the therapeutic strategy for FGFR3-TACC3 fusion gene positive glioblastoma,we first analyzed public cancer databases such as The Cancer Genome Atlas and Gene Expression Omnibus to evaluate the response of the FGFR3-TACC3 fusion gene positive glioblastoma to alkylating agent temozolomide.The FGFR3-TACC3 fusion protein not only maintains all activation and signal pathway transduction domains of wild-type FGFR3 but also has a stronger FGFR3 signaling pathway activity,so we evaluate the association between FGFR3 expression level and survival of glioblastoma patients.Additionally,we further analyzed the association between the FGFR3 gene expression level and the chemosensitivity of alkylating agents.In the in vitro section,we used the cell proliferation-toxicity detection kit and clonogenic assay to evaluate the sensitivity of glioblastoma cells stably expressing FGFR3-TACC3,wild-type FGFR3,kinase-inactivated FGFR3-TACC3,and empty vectors to alkylating agent temozolomide.To further investigate the mechanism of alkylating agents resistance conferred by FGFR3-TACC3,we applied immunoblot to detect the changes of FGFR3-TACC3 signaling pathways after long-term,high-concentration temozolomide treatment.Moreover,immunoblot and immunofluorescence staining were used to detect DNA damage marker p H2 AX in glioblastoma cells stably expressing FGFR3-TACC3,wild-type FGFR3,kinase-inactivated FGFR3-TACC3,and empty vector.To explore whether Hsp90 inhibitors enhance the sensitivity of glioblastoma cells to temozolomide by inhibiting FGFR3-TACC3 signaling pathways,we first set up a drug concentration matrix of temozolomide and Hsp90 inhibitor.The survival rate of tumor cells was measured using the cell proliferation-toxicity detection kit,and the drug combination effect index was analyzed using Calcu Syn 2.0.Then immunoblot and immunofluorescence staining were used to detect p H2 AX to evaluate the DNA damage by temozolomide and Hsp90 inhibitor in FGFR3-TACC3 positive glioblastoma cells.In the in vivo study,with the aid of the stereotaxic instrument,U-251 MG cells stably expressing FGFR3-TACC3 infected with luciferase virus were intracranially injected to establish a model of intracranial tumors in nude mice.The experimental group was administered with temozolomide(5mg/kg/day)by oral gavage,Hsp90 inhibitor Onalespib(30mg/kg/day)by tail vein injection and the combination of the two.Bodyweight and survival of tumor-bearing nude mice were monitored daily.Intracranial tumor size was monitored every 7 days using the in vivo imaging system.Tumor-bearing nude mice were sacrificed according to the body condition of the mice.Brain tumor tissue slices were stained for immunohistochemistry.Results: 1.FGFR3-TACC3 forms a ternary complex with Hsp90 and Cdc37.Inhibition of either Hsp90 or Cdc37 disrupts the formation of the ternary complex.2.FGFR3-TACC3 associates more Hsp90 and Cdc37 compared to wild type FGFR3.3.Inhibition of Hsp90 chaperone system abrogates the glycosylation of FGFR3-TACC3 fusion protein.4.Inhibition of Hsp90 chaperone system induces ubiquitination-mediated degradation of the glycosylated FGFR3-TACC3 fusion protein,resulting in inactivation of the FGFR3-TACC3 fusion protein.5.FGFR3-TACC3 is a strong Hsp90 client6.Onalespib and 17-AAG significantly inhibit the proliferation and colony formation of glioblastoma cells harboring the FGFR3-TACC3 fusion gene.7.FGFR3-TACC3 fusion protein confers resistance to alkylating agent temozolomide via the FGFR signaling pathway in glioblastoma.8.Combination therapy with Hsp90 inhibitor sensitizes temozolomide treatment in glioblastoma cells harboring the FGFR3-TACC3 fusion gene.Conclusions: The maturation process and glycosylation of nascent FGFR3-TACC3 fusion protein are accomplished by the Hsp90 chaperone system.The glycosylated FGFR3-TACC3 fusion protein maintains stability and phosphorylation activation under the chaperoning of the Hsp90 chaperone system.Inhibition of the Hsp90 chaperone system abrogates the glycosylation of the FGFR3-TACC3 fusion protein and induce ubiquitination-mediated degradation of the glycosylated FGFR3-TACC3 fusion protein.Hsp90 inhibitors significantly inhibit the phosphorylation activation of the FGFR3-TACC3 fusion protein and significantly inhibit the proliferation of glioblastoma cells harboring the FGFR3-TACC3 fusion gene.Combination therapy with Hsp90 inhibitors sensitizes temozolomide treatment in glioblastoma cells harboring the FGFR3-TACC3 fusion gene.