The Mechanism Of Transcriptional Factor Bach1 Regulating Glioblastoma Growth By Inhibiting Cyclin D1 And MMP2

Glioblastoma multiforme(GBM;World Health Organization grade IV glioma)is the most common and most aggressive brain tumor.GBMs can be classified into two categories,on the basis of the history of tumor onset.More than 90%of GBMs present as de novo GBMs(primary GBMs),and the remainder progress from low-grade(grades I–III)gliomas(secondary GBMs).The incidence increases with age,with the peak incidence being in the fifth or sixth decade.Current treatment regimens for newly diagnosed GBM are based on the randomized prospective trial,led by Roger Stupp,comparing external beam fractionated radiation alone to concurrent daily low-dose temozolomide during radiation,a one-month break,and then six months of adjuvant temozolomide at 150–200 mg/m~2.This trial demonstrated an increase in median overall survival of approximately three months and 30%versus 10%survival at 24months favoring the chemotherapy cohort.The overall survival for patients with newly diagnosed GBM remains short.Therefore,identifying alternative therapeutic approaches is critical.BACH1(BTB and CNC homology 1,basic leucine zipper transcription factor 1)is a transcriptional factor and a member of cap‘n'collar(CNC)and basic region leucine zipper factor family.In contrast to other bZIP family members,BACH1 appeared as a comparatively specific transcription factor.Since the discovery of Bach-1,in-vitro studies have proven the important information on the function of this gene in the development of migration and invasion in cancerous cells.Studies proved that BACH1is involved in bone metastasis of breast cancer by up-regulating metastatic genes like CXCR4 and MMP1.Future research is also expected to provide more insight into the Bach1 functions as a cancer migration and invasion factor.The investigation of involved genes in cancermigration and invasion progression and their signaling pathway is critical for understanding their predictive and therapeutic potential.The role of Bach1in cancer progression remains contentiously and the function of Bach1 in glioblastoma has not been identified.Therefore,the role of Bach1 in different caner needs to be further clarified.The main results are listed as follows:1.Bach1 is down-regulated in GBM and low expression of Bach1 correlates with poor patient prognosisTo better understand the role of Bach1 in GBM,we demonstrated that Bach1expression levels correlate with progressive stages of glioma by performing immunohistochemical staining(IHC)using primary tissue microarray samples from glioma patients.Bach1 expression was significantly decreased in tumor samples from patients with advanced stages of glioma compared with normal tissues from individuals with no cancer.Similarly,the expression of Bach1 was down-regulated in GBM cells compared with the normal glia cells.To determine whether alterations at the genetic locus of Bach1 could be implicated in GBM patient prognosis,survival data from TCGA database were used to evaluate the effects of Bach1 on overall patient survival.The low expression of Bach1 was very significantly correlated with reduced patient survival in TCGA's data.These results suggestes that Bach1 plays role as a tumor suppressor in glioblastoma.2.Bach1 suppresses GBM cell proliferation,metastasis and brain tumorigenesisTo determine the role of Bach1 in GBM cell proliferation,we generated a group of GBM cell lines with stable overexpression of Bach1.MTT assays with GBM cells demonstrated that Bach1 forced overexpression resulted in a significant inhibition in cell viability and that Bach1 knockdown led to a marked increase in cell viability.Furthermore,Bach1-overexpressing cells showed over a 35%reduction in DNA synthesis compared to control cells according to BrdU staining.Above results revealed that Bach1 could suppress GBM cell proliferation,similar results were obtained from colony formation assay.To provide further insight into the biological effect of Bach1,we evaluated cell cycle progression by flow cytometry in U87 and U251 GBM cells.We found that Bach1 forced overexpression induced cell cycle arrest at G1 phase.Then,we investigated whether Bach1 could also affect GBM cell metastasis.Representative pictures and their quantification showed that Bach1 was able to strongly inhibit cell migration and invasion.Conversely,when we silenced Bach1 endogenous levels with stable shRNA,we observed an increase of GBM cell metastasis.To investigate the role of Bach1 in GBM tumorigenesis,we injected U87 cells harboring an empty vector and U87 cells with Bach1 overexpression subcutaneously into athymic nude mice.We found that Bach1 overexpression resulted in significantly reduced tumor growth compared with tumors derived from control cells.Moreover,the above cells were intracranially injected into nude mice.Overexpression of Bach1significantly reduced the growth of brain tumors and prolonged the survival time of mice.These results together suggest that Bach1 is down-regulated in GBM and suppresses GBM progression.3.Bach1 directly inhibits the transcriptions of cyclin D1 and MMP2 in GBM cellsTo explore the underlying mechanism that Bach1 represses GBM progression,we detected some key regulator of cell cycle and metastasis.We found that cyclin D1 and MMP2 are the downstream targets of Bach1.To determine whether Bach1 directly inhibits the transcription of cyclin D1 and MMP2,we generated a series of luciferase reporter constructs containing cyclin D1 promoter and MMP2 promoter,then investigated the effect of Bach1 overexpression on the luciferase activity of these constructs.Overexpression of Bach1 significantly reduced the luciferase activity of cyclin D1 and MMP2 luciferase reporter constructs.To further explore whether Bach1directly binds to the promoter region of cyclin D1 and MMP2,we employed chromatin immunoprecipitation(ChIP)assay.The results indicated that Bach1 occupies the region from position–183 to–1 of the cyclin D1 promoter and the region from position–232to–1 of the MMP2 promoter in U87 cells.Cyclin D1 contains a consensus TCF/lef binding site from positions–177 to–171.MMP2 contains a consensus TCF/lef binding site from positions–224 to–218.Bach1 overexpression failed to reduce the activity of the promoter/reporter constructs when TCF/lef site were mutated.These results together suggest that Bach1 directly inhibits the transcriptions of cyclin D1 and MMP2 by binding to the TCF/lef site of their promoter.4.Bach1 plays roles in transcriptional repression and GBM progression dependent on BTB domainAs a binding protein,Bach1 can not bind to promoter directly.We find that only TCF4 can bind to the TCF/lef binding site of cyclin D1 and MMP2 promter through big data analysis.We detected Bach1 after immunoprecipitation of endogenous TCF4 from lysates of GBM cells,the results showed that Bach1 could interact with TCF4 in GBM cells.Also,exogenous Bach1 coprecipitated with TCF4 from the lysate of HEK293FT cells.We generated different vectors coding the full Bach1 sequence or the deletion sequences lacking either bZip domain or BTB domain to detect which domain plays role in transcriptional repression of cyclin D1 and MMP2.We found that TCF4coimmunoprecipitated with deleted Bach1 sequence,which contains BTB domain but not bZip domain.Moreover,dual-luciferase reporter assay shows that Bach1 the transcription of cyclin D1 and MMP2 dependent on BTB domain.According to MTT assay and transwell assay,we found that only Bach1-?bZip overexpression suppressed proliferation and invasion of GBM cells.All the results suggest that Bach1 interacts with TCF4,suppresses transcription and inhibits GBM progression dependent on BTB domain but not bZip domain.5.Activation of canonical Wnt signaling rescues the effects induced by Bach1overexpression in glioblastoma.Previous study shows that Bach1 induces repression of angiogenesis by inhibiting Wnt targets IL-8 and VEGF.Activation of canonical Wnt signaling can rescue this effects.Cyclin D1 and MMP2 are the downstream targets of canonical Wnt signaling.Therefore,we detect the effect of Wnt on Bach1 in glioblastoa.When canonical Wnt signaling was stimulated by Wnt3a treatment in Bach1-overexpressing cells,the ability of proliferation and metastasis was rescued.The activation of Wnt signaling significantly reduced Bach1/TCF4 coprecipitation without altering Bach1 expression.ChIP assay and dual-luciferase reporter assay show that the transcriptional repression induced by Bach1 overexpression was abrogated.These results suggest that activation of canonical Wnt signaling disrupts the binding of Bach1 to the promoter cyclin D1 and MMP2,rescues the effects induced by Bach1 in glioblastoma cells.6.Bach1 is a direct target of miR-155-5p in glioblastoma.In order to identify the underlying mechanism of deficiency in Bach1 expression,we put the focus on microRNAs(miRNAs).Based on bioinformatics analysis and clinic database,we find that Bach1 is a potential target of miR-155-5p in glioblastoma.To verify this prediction,the Bach1 3?UTR containing the miR-155-5p binding site was cloned downstream of luciferase ORF.This reporter construct was transfected HEK-293FT cells together with miR-155-5p mimics or NC,the relative luciferase activity of the reporter was inhibited by the mimics;however,there is significant change in the luciferase activity of mutagenesis reporter.Overexpressed miR-155-5p by transfecting mimics in GBM cells,we found that proliferation and invasion of GBM cells were increased.To determine whether Bach1 is a functional target of miR-155-5p,we generated stable U87-miR-155 overexpressing cells and U87-miR-155overexpressing cells with reconstituted expression of Bach1-?bZip or Bach1-?BTB.MTT assay showed that cell proliferation was promoted by miR-155 overexpression,and the promotion was abrogated by reconstituted expression of Bach1-?bZip but not of Bach1-?BTB.These results indicate that miR-155-5p directly inhibit Bach1,and then promote GBM progression,which can be abrogated by reconstituted expression of Bach1 with BTB domain.