Study On Clinical And Molecular Pathological Features Of Glioblastoma And The Molecular Mechanism Of NTSR1 Promoting Proliferation

Glioblastoma is the most common malignant brain tumor in adults with a poor prognosis.At present,the prognosis of glioblastoma patients has been improved by surgery,neoadjuvant chemotherapy and tumor-treating fields,but the median overall survival is 20.5 months.Therefore,it needs to develop new therapeutic targets,and effective treatment depends on our understanding of its proliferation and invasion mechanisms.Therefore,it is still a hot and difficult point to explore the mechanisms of glioblastoma proliferation and invasion.WHO classification of central nervous system tumors(4th revised edition)includes the rare subtypes of glioblastoma,such as epithelioid glioblastoma(E-GBM),and classifies glioblastoma into IDH wild type glioblastoma(GBM IDH-wt)and IDH mutant glioblastoma(GBM IDH-mut)depend on whether the IDH gene is mutant or not.We selected 138 cases with complete pathological data from 498 cases of glioblastoma,including 15 cases of E-GBM,and found 10 cases of GBM IDH1-mut from 165 cases of glioblastoma with IDH1 immunohistochemical results.The clinical pathological characteristics and prognosis factors of glioblastoma were summarized retrospectively,by pathological and molecular pathological analysis.As a neuropeptide widely is expressed in the central and peripheral nervous system,neurotensin(NTS)activates the downstream signal pathway through neurotensin receptor1(NTSR1).NTS/NTR1 signaling promotes glioma proliferation,migration,and invasion.NTSR1 is correlated with the ?-catenin/Tcf transcription complex Wnt/APC and directly targets cancer gene signal pathway,while another study found that NTS was direct target of Wnt/beta-catenin signaling pathway.We explored relationship between NTSR1 and molecular subtype of glioblastoma,NTSR1 and Wnt/?-catenin signaling pathway.Our study investigated the molecular mechanism of glioblastoma proliferation,provided new targets and strategies for individualized treatment of glioblastoma.Therefore,this study was divided into the following two parts.In the first part,clinical data of 138 cases of glioblastoma were summarized.The expressions of IDH1,ATRX,P53,EGFR,PTEN,CD44 and CHI3L1 were detected by immunohistochemical staining,and their molecular subtype and prognostic factors were analyzed.A retrospective clinicopathological study was conducted on 10 cases of GBM IDH1-mut and 15 cases of E-GBM.Fifteen cases of non E-GBM were randomly selected as a control,and histopathological observation,immunohistochemical staining and molecular pathology detection by En Vision method were performed to analyze the prognostic factors,in order to investigated the clinicopathological characteristics and prognostic factors of glioblastoma.In the second part,the molecular mechanism of NTSR1 promoting the growth of glioblastoma was detceted.Firstly,the expression of NTSR1 was detected in glioblastoma samples,and the correlation between NTSR1 expression and overall survival of glioblastoma patients,important molecular markers IDH1,ATRX and P53 and the prognosis were analyzed.We found that patients with NTSR1 expression had worse prognosis than those negative.Investigate the effects of NTS/NTSR1 on Wnt/?-Catenin pathway,by knockdown NTSR1 in two glioblastoma cell lines(A172 and U87),and overexpression of NTSR1 in U251 cell.We further investigate the regulatory mechanism of NTS/NTSR1 on Wnt/?-catenin signals and whether Wnt/?-catenin signals are involved in regulation of NTSR1,and finally evaluate the therapeutic potential of target on the NTSR1/Wnt/?-catenin loop.The main experimental results and conclusions are as follows:Part I results1.Clinical features:1.1 The average age of 138 patients was 61.4 years old,with a median age was 53 years old.There were 74 males and 64 females.Male: female was 1.16:1.Supratentorial cerebral hemisphere was predominant and subatentorial was rare.The frontal and temporal lobes were involved most frequently.Two lobes were involved in 26.08% of the patients.Dizziness,headache and vomiting were the main symptoms.23.19% of the patients relapsed,with median PFS 6.0 months.Median overall survival(OS)was 9.0 months,mean OS was 15.6 months.1.2 There were 15 cases of E-GBM,including 12 males and 3 females,with an average age of 39.6 years and a median age of 34 years.There were 15 cases of non E-GBM,including 11 males and 4 females,with an average age of 57.3 years and a median age of 63 years.There was a significant statistical difference between median age(p=0.002).There was no significant statistical difference in gender(p=1.000)and median OS(p= 0.079).1.3 There were 10 cases of IDH1 mutant GBM,including 6 males and 4 females,with an average age of 44.6 years.7 patients were involved in the frontal lobe,with dizziness and headache as the main symptoms,and 5 patients had a history of glioma.2.Histopathology: the common morphological features of GBM IDH1-mut and non E-GBM are cellular pleomorphism,atypia,brick mitosis,microvascular hyperplasia,palisading necrosis and coagulative ischemic necrosis.E-GBM were composed of consistent epithelioid cells and some rhabdoid cells,atypia,brick mitosis,microvascular hyperplasia,and mainly zone necrosis.Necrosis was divided into extensive areas and focal areas according to involved extent.3.Immunohistochemical characteristics3.1 Among 138 GBM patients,expression of IDH1 was positive in 9 cases,expression of EGFR was positive in 46 cases,expression of CHI3L1 was positive in 66 cases,ATRX deficient was found in 27 cases and expression of P53 was in 52 cases.3.2 GFAP,Nestin and IDH1 were positively expressed in 10 cases of GBM IDH1-mut,olig-2 was expressed variable(9/10),and P53 was diffusely expressed(9/10).ATRX deficient was in all cases.3.3 GFAP was positively expressed in 9 cases of E-GBM,positive focally in 6 cases of E-GBM.Vimentin,Nestin,s-100,c-met,INI1 and ATRX were expressed in 15 cases,and P53 was expressed in 7 cases.EMA and EGFR were focally positive in 6 cases,and CHI3L1 was focally positive in 4 cases.EGFR was focally positive in E-GBM(6/15),whileEGFR was diffusely expressed in nonE-GBM(10/15).Expression of EZH2 was in E-GBM(86.7%,13/15),overexpression of EZH2 was in E-GBM(60.0%,9/15);expression of EZH2 was in non E-GBM(86.7%,13/15),while overexpression of EZH2 was in non E-GBM(53.3%,8/15).There was no significant statistical difference in overexpression of EZH2(p=0.713)between E-GBM and non E-GBM.4.Molecular characteristics:4.1 The molecular subtype of 138 cases of glioblastoma were as follows: 66 cases were mesenchymal type,46 cases were classical type,9 cases were proneuronal type,and 17 cases were other/unclassified type.4.2 First-generation sequencing(Sanger sequencing)showed that IDH1R132 H mutation was observed in 10 cases of GBM IDH1-mut,IDH1R132 H mutation was not observed in 15 E-GBM cases,and IDH1R132 H mutation was observed in non E-GBM(1/15).There was no significant statistical difference in IDH1R132 H mutation(p=1.000)between E-GBM and nonE-GBM.4.3 MS-PCR showed that MGMT promoter was methylated in GBM IDH1-mut(9/10)and MGMT promoter was methylated in E-GBM(46.7%,7/15),MGMT promoter was methylated in non E-GBM(53.3%,8/15).There was no significant statistical difference in MGMT promoter methylation(p=0.715)between E-GBM and nonE-GBM.4.4 Quantitative Real-time PCR showed no BRAFV600 E mutation in GBM IDH1-mut(0/3),BRAFV600 E mutation in E-GBM(46.7%,7/15),and no BRAFV600 E mutation in non E-GBM(0/15).There was no significant statistical difference in BRAF mutation(p=0.01)between E-GBM and nonE-GBM.4.5 FISH showed codeletion of 1p/19 q in GBM IDH1-mut(0/3),no codeletion of 1p/19 q in E-GBM(0/15),and codeletion of 1p/19 q non E-GBM(1/15)cases.There was no significant statistical difference in codeletion of 1p/19q(p=1.000)between E-GBM and non E-GBM.There was no amplification of E-GBM EGFR in 15 cases.5.Prognostic analysis.5.1 Kaplan Meier curve analysis showed that gender(p = 0.823),P53(p=0.093)was no statistically significant correlation with OS.Univariate analysis showed that age(p = 0.007),IDH1(p = 0.013),ATRX(p = 0.023),molecular subtype(p = 0.019),treatment pattern(p = 0.000)were significantly associated with OS.Multivariate analysis showed that molecular subtype(p = 0.008),treatment pattern(p = 0.000)were statistically significant associated with OS.5.2 Six E-GBM patients with OS(?12 months)presented extensive necrosis(6/6),overexpression of EZH2(6/6),MGMT promoter unmethylated(5/6),BRAFV600 E mutation(3/6),treatment(surgery only,4/6).Three E-GBM patients with OS(>12 months)presented with focal necrosis(3/3),negative or low expression of EZH2(3/3),MGMT promoter methylated(2/3),BRAFV600 E mutation(0/3)and the treatment pattern(surgery + radiotherapy/chemoradiotherapy,2/3).5.3 Prognosis of GBM IDH1-mut patients: all the 4 patients with poor prognosis had significant angiogenesis,with an average density of 19.9/20 HPF and extensive necrosis.Among the 4 patients,two-lobe involved in 3.In 5 patients with favorable prognosis,tumor angiogenesis was mild,the average density was 10.1/20 HPF,and focal necrosis was observed,4 patients were all involved with one lobe.Part I conclusions:1.Glioblastoma patients' OS was related to molecular subtype,important molecular markers such as IDH1 and ATRX mutation,treatment pattern.Molecular subtypes and treatment pattern are independent prognostic factors for glioblastoma.2.E-GBM was rare and the prognosis was poor.Extensive necrosis,unmethylation of MGMT promoter,overexpression of EZH2 and lack of adjuvant chemoradiotherapy suggest poor prognosis of E-GBM.3.GBM IDH1-mut is rare.Tumor involved area,necrosis extent and microvascular hyperplasia density,treatment pattern are prognostic factors for patients with GBM IDH1-mut.Part 2 results1.Among 138 cases of GBM,80 cases were positive for NTSR1,and there was a statistically significant difference between the expression of NTSR1 in the GBM and molecular subtype(p=0.003).The positive rates of classic and mesenchymal subtypes were 67.4% and 63.6%,respectively,and the proneuronal and unclassificated subtypes were 11.1% and 35.3%,respectively.The expression of NTSR1 was negatively correlated with IDH1(p=0.001),and were positively correlated with ATRX(p=0.043).There was no significant statistical correlation between the expression of NTSR1 and P53(P= 0.263).Kaplan-meier curve analysis showed OS was a statistically negative correlation with NTSR1 expression,and univariate Cox analysis showed that patients with expression of NTSR1 had a worse prognosis than those without expression of NTSR1(p=0.008).2.NTSR1 knockdown and the treatment with NTSR1 antagonists SR48692 in A172 and U87 cell lines,the activity of Wnt/?-catenin pathway were significantly markedly reduced and m RNA expression of the downstream targets of Wnt/?-catenin(MYC,CCND1 and MMP7)were significantly markedly reduced,the phosphorylation levels of NF-?B and MAPK were markedly decreased.All the Wnts detected largely compromised by NF-?B inhibitor(TPCA-1)and MAPK inhibitor(U0126).Overexpression of NTSR1 in U251 cells significantly increased the activity of Wnt/?-catenin pathway and increased the expression level of downstream targets of Wnt/?-catenin.Wnt activator(Wnt3a)increased NTSR1 mRNA and NTSR1 protein levels,while Wnt inhibitor iCRT3 reduced the level of NTSR1(p<0.05).3.In vitro,proliferation rates of A172 and U87 cells were markedly upregulated by NTS or Wnt3a;and the prolific effects of NTS and Wnt3 a were largely compromised by combined treatment with SR48692 or i CRT3(p<0.01).Cell apoptosis of A172 and U87 cells was reduced by NTS or Wnt3 a treatment;and the anti-apoptotic effects of NTS and Wnt3 a can be largely abrogated by SR48692 or i CRT3(p<0.01).4.A subcutaneous xenograft model in NOD-SCID BALB/c mice showed the tumor growth rates were markedly retarded by SR48692 or iCRT3 treatment,accompanied with a reduction in Ki67 index,a proliferation marker(p<0.05).Part II conclusion1.NTSR1's expreesion was related to the molecular subtypes.NTSR1 expression was a factor of poor prognosis in glioblastoma.2.In glioblastoma,NTS/NTSR1 increased the activity of Wnt/?-catenin signal pathway by up-regulating the expression of NF-?B and MAPK.NTSR1 was regulated by Wnt/?-catenin pathway,and there was a positive feedback loop between NTS/NTSR1 and Wnt/?-catenin pathway.Targeted inhibition of NTSR1/Wnt/?-catenin loop significantly inhibited growth of glioblastoma cells.