The Studies On Regulation Function Of Transcription Factors GATA2 And FoxM1 On Glioma Malignant Phenotype

PartⅠ The role of transcription factor GATA2 in EGFR/ERK/Elk-1 signaling pathway of gliomasBackground and objective Epidermal growth factor receptor(EGFR) promotes cell proliferation and maintains physiological homeostasis by mediating activation of downstream molecules, such as ERK and Elk-1. However, overexpression of EGFR disturbing cell division and differentiation has a significant association with tumor progression, invasion and metastasis. Transcription factor GATA2, one of members of zinc finger structure GATA family, has two zinc fingers, by which GATA2 binds to the target gene, Elk-1, and induces its transcription. Overexpression of GATA2 was identified in many malignancies, including leukemia, breast cancer, and prostate cancer. The function of GATA2 in glioma remains to be clarified.Methods Establishment of GATA2 gene overexpression and knockdown glioma cell lines were performed by eukaryotic expression vectors and siRNA transfection in SW1783 and U87 cells. Immunoblotting was used to detect the expression levels of GATA2, EGFR, and Elk-1 in stable cell lines. Cell proliferation of overexpression and knockdown cells were determined using MTT assay. Cell invasion and metastasis were examined by Transwell assay. Elk-1 luciferase reporter assay was used to analysis the effect of GATA2 on Elk-1. Expression of GATA2 was determined in fifty-six glioblastoma multiforme(GBM) tissues and 6 controls.Results The results showed GATA2 overexpression promoted cell proliferation, invasion, and metastasis, whereas GATA2 knockdown inhibited these processes in SW1783 and U87 cells. The expression of GATA2 and its target Elk-1 decreased significantly after treatment with EGFR inhibitor AG1478 and ERK inhibitor U01226 in U87 cells. Furthermore, EGF induced expression level of GATA2 and its target Elk-1 in SW1783 cells. The results of immunohistochemistry staining showed GATA2 positive frequency is 73.2%(41/56) in GBM tissues compared with 16.7(1/6) in normal control tissues, which indicated the expression level of GATA2 is in negative correlation with patients survivals.Conclusions Overexpression of GATA2 was determined in glioblastoma multiforme tissues, which is an indicator of poor prognosis. GATA2 plays important roles in tumor proliferation, invasion, and metastasis regulated by EGFR/ERK/Elk-1 pathway, which remains potential to be a novel therapeutic target in glioma. Part ⅡThe role of transcription factor FoxM1 in Wnt/β-catenin signaling pathwayBackground and objective Previous studies show transcription factor FoxM1 is a novel downstream gene of the Wnt pathway. Fox M1 activates classic Wnt pathway by interacting with β-catenin to maintain self-renewal of neural stem cells. In addition, expression level of FoxM1 is higher in advanced GBM than in low-level astrocytomas. Several inhibitors, including IWP2 and LGK974, have been developed to target Wnt pathway in tumor therapy. As a novel regulator of Wnt pathway, Wnt-C59 blocks the function of Wnt by selectively inhibiting Porcupine, a member of MBOAT family, mediated Wnt secretion. The anti-tumor effect of Wnt-C59 has been proved in breast cancer. The role of Wnt-C59 in brain tumor is still unclear. This section is to determine the effect on glioma after inhibition of Wnt pathway.Methods Human glioma cell line U87 and human glioma stem cell line GSC11 were cultured and treated with exogenous Wnt3 a and inhibitor C59, respectively, to enhance and inhibit the expression of FoxM1. The expression levels of β-catenin, FoxM1, Axin-2 and Cyclin D1 of Wnt signaling pathway in glioma cell lines were investigated by immunoblotting(Western Blot). The effect of C59 on the activity of Wnt in glioma cells was detected by dual luciferase reporter gene(Top-FLASH) system. The changes of related downstream genes Cyclin D1 and C-myc after hindering the Wnt pathway and FoxM1 were examined by Real-time quantitative PCR. The effect of C59 on the cellular localization and nuclear import of β-catenin was investigated by cell immunofluorescence staining. The inhibiting effect of C59 to self-renewal ability of glioma stem cell was detected by Neurosphere Formation assay. The effect of C59 on abilities of migration and invasion of glioma cells was detected by Wound scratch assay and Transwell assay. Mapped growth curve by CCK-8 assays was investigated about the effect of TMZ and C59 on the proliferation of glioma cells.Results Immunoblotting(Western Blot) showed Wnt3 a and C59, could respectively, enhanced and inhibited the expression of FoxM1/β-catenin in glioma cells U87 and GSC11. C59 was proved to down-regulate the activity of Wnt signaling pathway and its downstream genes Axin-2 and Cyclin D1 in glioma cells significantly. Dual luciferase reporter gene(Top-FLASH) systems analysis showed C59 could significantly inhibit Wnt activity in glioma cells. The result of Realtime PCR indicated that C59 can result in the low-expression of Wnt-related gene Cyclin D1 and C-myc. The result of immunofluorescence staining technique showed C59 to prevent β-catenin translocating into the nucleus, expression level of β-catenin in nucleus decreased. Wnt inhibitor significantly inhibited self-renewal and the neurosphere formation of glioma stem cells. The wound scratch and transwell assays confirmed C59 can reduce the migration and invasion of U87 cells. C59 potentiated the cytotoxicity of TMZ in glioma cells. By combination with C59, TMZ inhibited cell proliferation of glioma with the dosage of 25μmol/L significantly.Conclusions For the first time, C59-Wnt pathway inhibitor was identified that it could significantly inhibit the expression of FoxM1 and β-catenin in glioma cell line and glioma stem cells, which results in the inhibitory effects of cell proliferation, migration, invasion, self-renew and the neurosphere formation. It is extremely essential for further study to use the inhibitor to regulate the expression of target molecules assoicated with malignant glioma phenotype. Part ⅢThe in vivo effects of combination Wnt pathway inhibitor C59 and temozolomide in gliomasBackground and objective A growing evidences show that several signaling pathways are involved in the regulation of malignant progression of gliomas, which results in the limitation of a single target molecule treatment. Temozolomide(TMZ), as a clinical first-line drug for glioma, has limited effects on the survival of patients. Therefore, on the basis of in vitro experiments this part proposed combination treatment of Wnt signal inhibitor C59 and TMZ on glioma in vivo experiments to investigate the feasibility of signaling pathway targeted therapy combination with the conventional chemotherapy program.Methods The brain glioma-bearing mouse model was established by stereotactic injecting glioma cells into the right striatum of adult male BALB/C mice. Nude mice were anesthetized after intraperitoneal injection with chloral hydrate, then make a 0.5cm long skin incision on the top of the mouse head, exposed skull, draw 10μl cell suspension(including U87 or GSC11 cells 5×105) with a micro syringe 25μl, injected cells slowly(5min) into the right caudate nucleus of mouse, and finished with bone wax closure holes. Animal model grouping and route of administration: experimental animals were divided into two large groups by inoculation of cell types(U87 and GSC11), in the U87 group and GSC11 group mice were divided into four groups according to therapy, the control group(no administration), C59 treatment group, TMZ treatment group, C59 combined TMZ therapy group. From the second day mice received treatment by group: no drugs, C59 gavage 10mg/kg/d for 14 days, TMZ gavage 50mg/kg/d for 14 days, C59 and TMZ combination gavage for 14 days. The tumor volume and immunohistochemistry: intracranial inoculation cells were treated with different groups of medications in each group, animals were sacrificed on the 28 th days after tumor implantation, brain tissue was collected, tumors were calculated(each group, n=5) by HE, and immunohistochemical staining to detect FoxM1 and β-catenin gene expression in xenografts. Tumor-bearing mice survival test: after intracranial inoculation of cells, mice were treated with different treatment groups(each group, n=10), each group were maintained to monitor the survival curves. Kaplan–Meier survival curves were plotted for each group.Results After successfully performed the intracranial xenografts mice model, nude mice had good survival state on early days. After 28 days two tumor-bearing mice appeared cachexia symptoms. On the 28 th day after intracranial tumour implantation, animals were sacrificed. It was found in all animal model of brain tissue tumorigenicity was 100%. After HE staining measurement, shortest diameter(a) and the longest diameter(b)of tumor, tumor volume was calculated according to the formula V=a2×b/2, results showed in the U87 group and GSC11 group, C59 and TMZ inhibited the tumor’s growth in nude mice, particularly in the combined treatment group,the treatment effect was most significantly. The survival of tumor-bearing mice: U87 group within the control group, C59 treatment group, TMZ treatment group, the median survival of C59 combined TMZ therapy group were 38, 54, 66, and 94 days, GSC11 group results were 32, 48, 52, and 85 days, suggesting that C59, TMZ could prolong the survival time of tumor-bearing mice, especially in the combination therapy group survival was significantly prolonged, compared with that of other groups. Immunohistochemical staining of FoxM1 and β-catenin gene showed, both C59 alone and combined with TMZ therapy could significantly decrease their level of expression in xenografts.Conclusions The efficacy of C59 combined TMZ therapy of intracranial glioma in nude mice bearing human glioma model, in terms of tumor volume and survival of tumor-bearing mice, was better than single C59 and single TMZ therapy, which provided a further exploration on the feasibility of the target molecule in combination with traditional chemotherapy treatment program.