The Role And Mechanism Studies Of Lysine Hydroxylase PLOD2 In Hypoxia-induced Glioma Migration And Invasion

BackgroundGlioblastoma multiforme(GBM,WHO grade Ⅳ)is the most common and fatal primary malignant tumor in the brain.Despite advances in surgical and medical therapy,the life expectancy of patients with GBM averages a mere 14 months following diagnosis.It is impossible to resect the whole mass because of GBM infiltration into the surrounding normal parenchyma,which is believed to be the main reason for the resistance of GBM to various therapeutics.Thus,investigation of molecular mechanisms driving tumor invasion is essential for the development of a curative therapy of the disease.To fully understand GBM invasion requires some understanding of the unique and complex tumor microenvironment as well as the tumor cell.A hallmark characteristic of the GBM microenvironment is hypoxia,and it is closely associated with tumor growth,progression and resistance to conventional cancer therapies.Hypoxia inhibites the degradation of hypoxia-inducible factor(HIF)-1/2α,which dimerizes with HIF-1β,the constitutively active subunit,to bind hypxia response element and drive gene transcription.How hypoxia regulates specific proteins involved in invasion might therefore help in the development of new therapies.The extracellular matrix(ECM)remodel is as a second characteristic change in the tumor microenvironment.During tumor progression,it has been indicated increased ECM stiffness,because of increased collagen deposition and cross-linking,promotes tumor cells migration and invasion.And it is commonly reported increased ECM stiffness in solid cancers,including glioma,and collagen,one of the major components in ECM,has been indicated to promote GBM angiogenesis and cell adhesion.The formation of collagen cross-links is initiated by procollagen-lysine 2-oxoglutarate 5-dioxygenase 2(PLOD2).Lysyl hydroxylase includes three isoforms--PLOD1,PLOD2 and PLOD3.PLOD1 is responsible for lysine hydroxylation in the a-helical or central domain,PLOD2 hydroxylates lysines in the telopeptide of procollagens,and isoform 3 specific substrate remains unclear.It is necessary to form collage cross-links that lysine residues are hydroxylated in telopeptide domian.Over-accumulation of collagen in fibrotic diseases is caused by overhydroxylation of the collagen telopeptides,as well as a large number of pyridinoline crosslinks formed by hydroxylated telopeptide lysines.These cross-links are difficult to be degraded by MMPs.PLOD2 is the only lysyl hydroxylase responsible to collagen cross-links.Interestingly,the role of PLOD2 has been reported in diseases,such as Bruck Syndrome and Ehlers-Danlos Syndrome type VIA,but not in glioma.Through histology-based expression profiling,PLOD2 has been identified as a novel prognostic marker in GBM.In the above context,based on prior results,this present study was to investigate the roles of PLOD2 biological effects and underlying molecular mechanisms in glioma at the clinical and cell levels via public databases,glioma specimens collecting,immunohistochemistry,picrosirius red staining,ELISA,lentiviral vectors infection and orthotopic xenografts models,in order to develop new and potential therapeutic targets for glioblastoma.Part Ⅰ Expression of PLOD2 in glioma tissuesObjectiveTo investigate the expression of PLOD2 in glioma tissues and study the clinic valueMethods1.Based on the Oncomine datasets,meta-analysis of PLOD2 mRNA expression level in patients with glioblastoma.2.Based on REMBRANDT Database,analyzed PLOD2 mRNA expression level in different grade gliomas.3.Immunofluorescence staining was performed to show PLOD2 expression level in different grade gliomas tissue biopsies.4.Based on TCGA Database,Kaplan-Meier analyzed overall survival(OS)and progression free survival(PFS)between GBM patients with different PLOD2 expression level.Results1.PLOD2 expression is positively correlated with tumor gradeBased on the Oncomine database,a meta-analysis of 7 independent GBM datasets,consisting of 861 human GBM samples,revealed that PLOD2 was significantly and consistently overexpressed in GBM across all datasets(,Mean Difference IV Random:1.71;CI:1.23-2.19;P<0.001).Next,expression data from the REMBRANDT database for PLOD2(n=178)was shown that PLOD2 mRNA expression was found to be significantly increased(～3-fold;P<0.0001)in GBM samples,compared to normal brain,grade Ⅱ,Ⅲ gliomas.Immunohistochemistry demonstrated that PLOD2 was highly expressed in grade Ⅲ gliomas and to a greater degree in GBM,whereas staining was absent or weak and appeared in fewer cells in normal brain tissues and grade Ⅱ gliomas.The expression pattern of PLOD2 was dependent on tumor grade,with the exception that no significant difference was observed between normal brain tissues and grade Ⅱ gliomas.Besides,based on the data from TCGA and REMBRANDT databases,the receiver operating characteristic(ROC)curve was utilized to determine whether PLOD2 could be used as a biomarker to distinguish between non-GBM and GBM patients.The area under ROC curve was 0.785(CI,0.785-0.828),indicating that PLOD2 might be effective as a diagnostic marker to distinguish GBM from lower grade gliomas.2.PLOD2 expression level is negatively correlated with the prognosis of glioblastoma patientsKaplan-Meier estimated based on the TCGA dataset showed significant differences in overall survival(OS)and progression free survival(PFS)between GBM patients with low PLOD2 expression and those with high expression.The median OS of GBM patients with low PLOD2 expression was 14.95 months(95%CI,9.484-20.052),compared to 13.93 months(95%CI,12.884-15.020)among those with high expression.The median PFS for GBM patients with low and high PLOD2 expression was 10.22 months(95%CI,6.03-14.41)and 7 months(95%CI,6.141-7.859),respectively.Conclusion1.PLOD2 expression is positively correlated with tumor grade;2.PLOD2 expression is negativly correlated with the prognosis of glioblastoma patientPart Ⅱ Hypoxia induces PLOD2 expression in glioma cellsObjectiveTo explore potential mechanism of PLOD2 expression in glioma cellsMethods1.Western blot analysis was performed to examine PLOD2 expression from lysates of U87 and U251 cells under hypoxic conditions.2.Western blot analysis was performed to examine PLOD2 expression in U87 and U251 cells after knockdown of HiF-1α/2α of inhibitor treatment under hypoxic conditions.3.Correlation analysis between PLOD2 mRNA expression level and HIF-1α mRNA expression levelResultsHypoxia induces PLOD2 expression through HIF-1α in glioma cellsTo determine whether hypoxia induced expression of PLOD2 in human glioma cells,we examined PLOD2 expression at mRNA and protein level respectively after hypoxia treatment for 48h.Meanwhile,lysates prepared from cells incubated under hypoxic conditions for different time points(0 to 48 h)were examined by Western blot.PLOD2 protein levels increased in both U87 and U251 cells～2 to 3-fold in a time dependent manner under hypoxia,compared to cells cultured under normoxic conditions.To investigate whether HIF-1α or HIF-2α were transcriptional regulators of PLOD2 under hypoxia,siRNA knockdown experiments were performed.PLOD2 protein levels in U87 and U251 cells transfected with siHIF-1α were reduced under hypoxia as determined by Western blot.However,no changes in PLOD2 expression were observed under hypoxia with transfection of siHIF-2α.The role of HIF-1α in the regulation of PLOD2 was further investigated through exposure of glioma cells to the HIF-1α inhibitor PX-478.Inhibition of HIF-1α by PX-478 also led to reduced levels of PLOD2 protein in U87 and U251 cells under hypoxia.Furthermore,PLOD2 mRNA expression level was closely correlated to HIF-1α mRNA expression level(r2=0.215,P<0.0001)based on TCGA database.ConclusionHypoxia induces PLOD2 expression through HIF-1α in glioma cellsPart Ⅲ The Role and Mechanism of PLOD2 in Hypoxia-induced Glioma Migration and InvasionObjectiveTo explore the role and mechanism of PLOD2 in the hypoxia-induced glioma migration and invasionMethods1.CCK8 assasy and colony formation assays were employed to access cell viability after knockdown of PLOD22.ELISA was performed to examine collagen I concentration from surpernatant of U87 and U251 cells under hypoxic conditions.3.Transwell models were performed to access cell migration after knockdown of PLOD2 in glioma cells under normoxic and hypoxic conditions.4.3D invasion assays were performed to access cell invasion after knockdown of PLOD2 in glioma cells under normoxic and hypoxic conditions.5.Laser scanning confocal microscope revealed glioma cells phenotype changes after knockdown of PLOD2 under normoxic and hypoxic conditions.6.U251 Subcutaneous and orthotopic xenografts glioma models were adopted to evaluate the function of PLOD2 in vivoResults1.Decreased PLOD2 expression in glioma cells did not inhibit their proliferation and collagen contentTo study the functions of protein PLOD2 in glioma progression in vitro,we generated U87 and U251 subclones that were stably transfected with a control vector(shNC)or a shRNAs against PLOD2 vector(shPLOD2).The effect of PLOD2 on cellular proliferation was performed in U87 and U251 cells using Cell Counting Kit 8 assay and colony formation assays,which showed that the expression of PLOD2 did not influence cells proliferation,compared with the control groups.The overall collagen content form cells supernatant was measured via collagen I ELISA kit.The results were shown that hypoxia induced collagen I secretion,PLOD2 knockdown did not affect overall collagen I amounts in both shNC and shPLOD2 groups under normoxia or hypoxia.2.Decreased PLOD2 protein inhibits migration and invasion of glioma cellsControl(shNC)or PLOD2 knockdown(shPLOD2)cell suspensions were seeded into the upper chamber of an uncoated Transwell membrane insert,while the lower chamber was filled with medium(20%FBS),and then cultured under normoxic or hypoxic conditions.Although hypoxia promoted migration of controls(～1.5-fold,P<0.001),loss of PLOD2 inhibited migration in shPLOD2 expressing clones,particularly under hypoxic conditions(>30%,P<0.001).In 3D culture,U87 and U251 cells grew into more structurally well-organized spheres,and formed the longest invasive projections surrounding the spheres under hypoxic conditions.Knockdown of PLOD2 inhibited the distance cells invaded into the Matrigel under both conditions.3.PLOD2 promotes migration and invasion through FAK signaling in glioma cellsExpression level of MMPs can be performed to assess tumor cells migration and invasion.However,knockdown of PLOD2 did not lead to alterations in MMP-2 and MMP-9 protein levels under normoxic or hypoxic conditions in U87 or U251 cells.A second approach to evaluate migration and invasion is through characterization of cell morphology and cytoskeletal status.Laser scanning confocal microscope revealed that hypoxia significantly increased focal adhesions in glioma cells,as determined by increased co-localization of phalloidin-labelled actin fibers(red)with paxillin(green)in focal adhesion plaques,compared to cells under normoxia.Knockdown of PLOD2 inhibited formation of focal adhesion plaques and led to disorganization of actin stress fibers under both normoxia and hypoxia.In addition,glioma cells were exposed to the FAK inhibitor TAE226 under normoxia and hypoxia.Western blot confirmed that TAE226 inhibited phosphorylation of FAK(Tyr 397)under both normoxic and hypoxic conditions.And migration and 3D invasion of parental U87 and U251 were inhibited by treatment with TAE226.4.PLOD2 enhances glioma cell invasion in vivoSubcutaneous and orthotopic glioma xenografts models were performed to assess the function of PLOD2 in vivo.Tumor volume of subcutaneously inoculated U251－shNC and U251-shPLOD2 cells was similar among groups.Hydroxylproline levels,which were used as an indicator of collagen content,were not significantly different between groups.Stiffness measurements made with compression and indentation tests demonstrated that knockdown of PLOD2 reduced tumor stiffness(P<0.05).Pathological analysis of orthotopic xenografts revealed that U251-shNC cells generated tumors with irregular,locally invasive borders,whereas U251-shPLOD2 tumors were more circumscribed.Picrosirius red staining,which is used to highlight collagen crosslinking,confirmed that PLOD2 was active in tumors.Collagen in U251-shNC tumors(control)was more cross-linked than in U251-shPLOD2 tumors.Conclusion1.PLOD2 promotes hypoxia-induced migration and invasion through FAK signaling in glioma cells2.Knockdown of PLOD2 significantly inhibits glioma cells invasion in vivo...