The Role Of FOXM1 In Brain Glioma Growth And Drug Resistance

High-grade brain gliomas are one of the leading causes of cancer mortality in adults,and impose great challenge on clinical therapy.Owing to the introduction of alkylating agent temozolomide(TMZ)and adoption of the radiotherapy with concomitant and adjuvant temozolomide strategy,the median survival of patients with glioblastoma multiforme(GBM,WHO IV)improved from 12.1 to 14.6 months.However,the overall clinical effect of this regimen is still disappointing,mostly due to the inherent or induced resistance to TMZ.Thus,more comprehensive understanding of the progression and resistance mechanisms and relevant novel therapeutic targets are urgently needed for this fatal tumor.FOXM1 is upregulated in various human cancer tissues and involves in almost all the hallmarks of malignant cancer.In regard of brain gliomas,upregulated expression and contribution to glioma stem cell(GSC)and TMZ resistance of FOXM1 were also reported by a few studies.However,there is still urgent need of a systematic investigation about the roles and mechanisms of FOXM1 in brain glioma growth and TMZ resistance,as well as the novel therapy of targeting FOXM1.1.The roles of FOXM1 in brain glioma growth and TMZ resistance1.1 Analyzing the expression and biological function of FOXM1 in public databases.With the help of some data processing tools,we firstly analyzed the mRNA expression of FOXM1 in brain gliomas from TCGA,CGGA and RAMBRANDT.FOXM1 mRNA level was upregulated in glioma samples and increased with increasing glioma grade in all three databases.Survival analysis with Kaplan-Meier method showed that FOXM1 mRNA level was negatively related with overall survival(OS)when all glioma patients or lower grade glioma(LGG)patients,but not GBM patients were used as research object.Via cBioPortal for Cancer Genomicsdata platform,we selected genes that positively correlatedwith FOXM1 expression in LGG or GBM or in both from TCGA.Genes that positively correlated with FOXM1 in both LGG and GBM were under cellular location and biological function analysis via FunRich12.0.1.2 FOXM1 promoted the growth of brain glioma.We initially established FOXM1 overexpression U251 and U87 cell lines via lentivirus transfection.The overexpression efficacy was confirmed by reverse transcriptionquantitative real-time PCR(RT-qPCR),western blot and immunocytofluorescence(ICC)method.On the contrary,we also knocked down FOXM1 in LN229 cells via transfecting siRNA.MTT assay demonstrated much higher optical density(OD)values in FOXM1 overexpression cells and lower OD values in FOXM1 knockdown cells,comparing to their corresponding control cells.EdU-594 cell proliferation experiment also showed that FOXM1 overexpression promoted while FOXM1 knockdown retarded the proliferation of glioma cells.What’s more,the colonic formation and spheroid growth were also enhanced by FOXM1 overexpression and reduced by FOXM1 knockdown.In order to confirm these in vitro results,we further established in vivo glioma models in mice.In nude mice subcutaneous models,tumor volumes and weight in FOXM1 overexpression group were significantly higher than those in control group;In NOD-SCID mice orthotopic models,the surface area of tumors derived from FOXM1 overexpression U87 cells was much larger than that from control cells.These results indicated the important role of FOXM1 in glioma cell proliferation and tumorigenicity.1.3 FOXM1 contributed to TMZ resistance in brain glioma.MTT assay results showed that FOXM1 overexpression U251 and U87 cells were less sensitive to TMZ treatment than their control cells,while FOXM1 knockdown LN229 cells were much more sensitive to TMZ than control cells.γ-H2 AX was used to mark the DNA double break after TMZ treatment,ICC showed that FOXM1 overexpression groups had less γ-H2 AX expression than control groups,while siRNA transfected group had much intenser γ-H2 AX staining than its control.Flow Cytometry experiments also showed that FOXM1 overexpression reduced and FOXM1 knockdown enhanced the apoptosis-inducing effect of TMZ.The effect of FOXM1 overexpression on TMZ was further tested in xenograft models subcutaneously transplanted in nude mice.When under same dose TMZ treatment,the volumes and weight of tumor from FOXM1 overexpression cells weremuch higher than those from control cells.2.FOXM1 regulated Survivin transcription in brain glioma2.1 Survivin expression was positively correlated with FOXM1 expression.Via analyzing brain glioma datasest from TCGA,CGGA and RAMBRANDT with some bioinformatic tools and methods,we found that Survivin expression was significantly upregulated in glioma tissues,with higher mRNA level in higher grade gliomas.Survival analysis with Kaplan Meier method showed that Survivin mRNA expression was negatively related with patient OS in all three databases.Pearson correlation test showed that Survivin mRNA expression was positively correlated with FOXM1 mRNA expression.Especially in glioma datasets from TCGA,Survivin was found to be significantly upregulated in both LGG and GBM,and also positively related with FOXM1 expression in both LGG and GBM.Clustering Analysis in heatmap showed that,among all typical members of anti-apoptosis protein family,Survivin had the closest relation with FOXM1 expression.Then we measured the mRNA levels of FOXM1 and Survivin in six glioma cell lines by RT-qPCR and found they were significantly correlated with each other.2.2 Survivin was a direct target of FOXM1.RT-qPCR and western blot results showed that mRNA and protein levels of Survivin were upregulated in FOXM1 overexpression U251 and U87 cells and downregulated in siRNA transfected LN229 cell line.Similar results were also demonstrated by ICC method.In order to found out whether Survivin gene(BIRC5)was a direct target of FOXM1 or not,we initially got the most possible binding sequence of FOXM1 on BIRC5 promoter via online software LASAGNA-Search 2.0.Luciferase reporter gene experiment showed that FOXM1 overexpression cell lines transfected with reporter plasmid containing wildtype promoter sequence had the highest luciferase activity,while both FOXM1 overexpression and control cell lines transfected with reporter plasmid containing mutant promoter sequence had the lowest luciferase activity.Then we tested Survivin’ role in FOXM1 induced TMZ resistance.MTT assay showed that Survivin knockdown reduced TMZ resistance in both control and FOXM1 overexpression cell lines.Colony formation experiment showed that Survivin inhibitor YM155 significantly reduced the colonicformation ability of FOXM1 overexpression cells.Then RT-qPCR,western blot and ICC experiments showed that FOXM1 and Survivin expression were markedly upregulated in TMZ resistant glioma cell lines.2.3 FOXM1-Survivin axis was upregulated in clinical glioma samples and related to poorer prognosis.Via RT-qPCR and immunohistochemical(IHC)method,we detected the mRNA and protein expression of FOXM1 and Survivin in clinical glioma samples.We found that both mRNA and protein levels of FOXM1 and Survivin were upregulated in most glioma samples,and there were significantly positive correlations between FOXM1 and Survivin mRNA expression and protein expression.Survival analysis with Kaplan-Meier method further revealed the prognosis predicting roles of FOXM1 and Survivin protein expression in all glioma patients cohort and LGG patients cohort,but not in GBM patients.Chi-square tests in SPSS17.0 showed that FOXM1/ Survivin protein expression was significantly correlated with Karnofsky score(KPS),WHO grade and recur time(Interval).Both univariate and multivariate analysis with cox proportional hazards model showed that FOXM1 level and Survivin level were significant risk factors for patient prognosis.3.Bortezomib reduced growth and TMZ resistance of brain glioma viadownregulating FOXM1-Survivin axis3.1 Bortezomib downregulated FOXM1-Survivin axis.RT-qPCR and western blot results showed that mRNA and protein levels of FOXM1 and Survivin in U251 and U87 cells were significantly downregulated by 10 nM and 20 nM Bortezomib treatment for 48 hours;These results were further confirmed by ICC experiments,in which the fluorescence intensity of FOXM1/Survivin in treatment groups were markedly lower than that in corresponding control groups.The protein expression of FOXM1/Survivin in nude mice subcutaneous glioma models were detected by IHC staining,and we found that FOXM1 and Survivin intensity in Bortezomib group and Bortezomib+TMZ group were much lower than that in control group,while FOXM1 and Survivin intensity in TMZ group were much higher than that in control group.3.2 Bortezomib inhibited proliferation of glioma cells.MTT assay showed that Bortezomib inhibited the viability and proliferation of U251 and U87 cell lines in a dose and time dependent manner.The minimum efficient concentration of Bortezomib on U251 and U87 cells was 10 nM and 5nM respectively.The day2 and day4 IC50 of Bortezomib were also calculated.Flow Cytometry experiments showed that 10 nM and 20 nM Bortezomib induced obvious cell apoptosis in glioma cells,with significant increase in both early and late stage cell apoptosis;Flow Cytometry experiments also showed that Bortezomib induced obvious cell cycle arrest,which resembling a G1 phase arrest.The tumor cell spheroid growth and colony formation were also markedly inhibited by 10 nM and 20 nM Bortezomib treatment.After pre-treatment with10 nM and 20 nM Bortezomib or drug vehicle,glioma cells were replanted in medium for stem cell culture.Bortezomib pre-treated U251 and U87 cells had much less stem cell colonies/spheroids than corresponding control cells.3.3 Bortezomib reduced TMZ resistance and enhanced TMZ efficacy.MTT assay showed that 10 nM Bortezomib and 200μM TMZ combination led to much lower cell viability than Bortezomib or TMZ alone.In accord with this,Bortezomib and TMZ combination caused much smaller surface area in glioma cell spheroid growth assay.Flow Cytometry experiments also showed that Bortezomib and TMZ combination induced significantly higher cell apoptosis and much severer G1 arrest in U251 and U87 cells than Bortezomib or TMZ alone had done.In xenograft glioma models established subcutaneously in nude mice,tumor growth rate(tumor volumes)and final tumor weight in Bortezomib and TMZ combination group were much lower than that in Bortezomib group and TMZ group.In intracranial orthotopic glioma models transplanted in NOD-SCID mice,final tumor sizes were measured in HE(Hematoxylin and eosin)stained horizontal slices,average tumor size in combination group were much smaller than that in Bortezomib group and TMZ group.4.Interaction between FOXM1-Survivin axis and NF-κB pathway4.1 NF-κB regulated the expression of Survivin but not FOXM1.RT-qPCR and western blot results showed that mRNA and protein levels of FOXM1 were not significantly altered after treatment with NF-κB inhibitor Pyrrolidine dithiocarbamate(PDTC).Via ICC experiment,we further found that nuclear expression of NF-κB was not significantly altered by Bortezomib,so Bortezomib downregulated FOXM1-Survivin axis in a NF-κB independent way.However,expression of Survivin,BCL-XL and MGMT(O6-methylguanine-DNA-methyltransferase)were markedly reduced by PDTC.On the contrary,expression of Survivin,BCL-XL and MGMT were markedly increased by TMZ treatment,and the TMZ stimulated overexpression of BCL-XL and MGMT,but not Survivin,were abrogated by concurrent PDTC treatment.This indicated that TMZ also stimulated Survivin expression via other NF-κB independent pathways,such as FOXM1.4.2 NF-κB inhibitor PDTC sensitized U251 cells to TMZ treatment.In order to explore the possibility of a combined strategy with Bortezomib,TMZ and PDTC,we initially tested the efficacy of PDTC in U251 cells,alone and in combination with TMZ.MTT assay showed that PDTC inhibited the viability of U251 cells in a dose and time dependent manner.PDTC and TMZ combination led to much higher inhibition rates than PDTC or TMZ had done.Flow Cytometry experiments also showed that PDTC induced cell apoptosis and cell cycle arrest in a dose dependent manner.Even in a concentration as low as 20 nM,the addition of PDTC to TMZ treatment led to significantly higher percentages of cell apoptosis and much severer cell cycle arrest.In summary,our present study showed that FOXM1 enhanced the progression and TMZ resistance of brain gliomas.Classic anti-apoptosis protein Survivin was a direct target of FOXM1;FOXM1-Survivin axis was upregulated in brain gliomas and correlated with poorer prognosis in glioma patients.We further found that FOXM1-Survivin axis could be specially downregulated by proteasome inhibitor Bortezomib,which reduced the growth and TMZ resistance of brain glioma both in vitro and in vivo.The expression of Survivin but not FOXM1 was regulated by NF-κB,and its inhibitor PDTC might be a potential drug for combination therapy.