Mechanism And Role Of A Disintegrin And Metalloproteinase17in U87Glioma Stem Cells-mediated Migration And Invasion

Malignant gliomas, the most prevalent primary central nervous system tumor, account for approximately35.26%-60.96%(median44.6%) of all cases of malignant primary brain tumors that are diagnosed in adults each year. Despite positive surgery and conventional radio-chemotherapy, The prognosis for patients with glioma remains dismal. Moreover, the outcome of glioblastoma multiforme (GBM), the most aggressive and malignant glioma (WHO grade IV) is even worse, with a median survival of14months. This high mortality is attributed to the aggressive invasion from malignant glioma cells, which is closely related to tumorigenesis and tumor progression. Therefore it is necessary to further characterize the mechanisms involved in invasion by malignant glioma in order to diseover new therapeutic targets.Tumor microenvironment plays a crucial role in guiding the invasion ability of glioblastoma cells. Glioma cell invasion, a very multi-step process, involve the attachment of tumor cells to extracellular matrix (ECM), degradation of ECM components, and subsequent penetration into tumor blood vessels to adjacent brain structure. These processes are accomplished in Part by matrixmetalloproteinases (MMPs) and a-disintegrin and metalloproteinase (ADAMs), the families of extracellular matrix degradation enzyme systems and zinc-dependent proteinases. As an important member of the ADAM family, ADAM17, termed tumor necrosis factor-a converting enzyme (TACE), is the primary secretase responsible for releasing the soluble form of tumor necrosis factor-α (TNF-α) from the plasma membrane and primary sheddase for multiple epidermal growth factor receptor (EGFR) pro-ligands, hence activating downstream signal transduction pathways to promote tumor progression.Recent studies have found the expression and activity of ADAM17increase under some human tumor tissues, including non-small cell lung cancer, breast cancer, ovarian cancer, renal cancer, prostate and pancreatic cancer. Moreover the high expression of ADAM17has been confirmed on glioma cell line. Zheng et al established glioma cell line U87MG with low ADAM17expression by shRNA transfection and showed that knockdown of ADAM17significantly reduced the proliferation and invasiveness of U87MG. Glioma as a kind of solid tumor with characteristics of invasion and infiltration, relies on degradation of ECM components, attachment of tumor cells to ECM, rapid proliferation of tumor cell, and abundant blood vessels for its migration. ADAM17has been associated with the process of proteolytic shedding of transmembrane proteins and hence the rapid modulation of downstream signal transduction pathways in the tumor microenvironment, which associated with tumorigenesis and tumor progression. ADAM17is involved in proteolysis of collagen Ⅴ、Ⅶ、Ⅹ, gelatin and elastic fibers of Extracellular Matrix (ECM), the release of several integrins from the cell surface, suggesting that ADAM17affects tumor angiogenesis and the invasive activity of glioma cells; Tumor angiogenesis is regulated by aberrant production of proangiogenic and antiangiogenic factors produced by malignant tumor. The expression and activity of ADAM17are important in modulating vessel development and promoting the expression and secretion of angiogenic factors, such as VEGF/VEGFR, HIF, bFGF, chemokines, EGF/EGFR, interleukins. They promote endothelial cell proliferation, migration and tubule formation, which are cruiel steps in angiogenesis and tumor invasion; Moreover, ADAM17is a primary sheddase for multiple epidermal growth factor receptor (EGFR) pro-ligands, including tumor necrosis factor-alpha (TNF-a), transforming growth factor-alpha (TGF-α), amphiregulin, interleukin-6, L-selectin, and amyloid precusor protein (APP). EGFR ligand-binding induces receptor phosphorylation, and subsequently activates downstream PI3K/AKT and MEK/ERK pathways, which contribute to invasiveness, angiogenesis and other malignant phenotypes of glioma. A better understanding of the roles of ADAM17and downstream EGFR signalling pathway on glioma invasion will help in developing therapeutie strategies to decrease the invasion of a malignant gliomas.Cancer stem cells (CSCs), or tumor initiating cells, open a new perspective for targeted therapy against glioma because of their special biologic behaviors and the important roles in tumorigenesis. Accumulating evidence suggests the existence of a small reservoir of glioma cells that share similar properties with neural stem cells (NSCs) and are capable of driving tumorigenesis. These cells, termed glioblastoma stem cells (GSCs) with the potential for extensive proliferation, self-renewal and multilineage differentiation, are more resistant to chemo-and radiotherapy, playing an important role on glioma initiation, growth and recurrence. Eliminating GSCs seems to be a new and prosperous therapeutic strategy for glioblastoma.The great progress of study on GSCs is enormously challenging traditional theory of tumor invasion. For instance, whether and how GSCs mediate tumor invasion. Our previous results showed that GSCs expressed higher levels of ADAM17compared to non-GSCs cells. Besides, recent studies have showed that EGFR signalling pathway plays critical role for the maintenance of biological properties in GSCs, leading us to speculate that ADAM17, the primary upstream component of multiple EGFR pro-ligands, possibly contributes to the invasive ability of GSCs. This study was based on our hypothesis that ADAM17was involved in GSCs-mediated glioma invasion by regulating EGFR signalling pathway.In the current study, we investigated the expression of ADAM17in glioma tissues of different malignancy and its correlation with patients outcome. Positive correlation between the expression levels of ADAM17and malignancy of gliomas was observed. There was a highly significant difference in overall survival between ADAM17high expression and low expression patients, with poorer outcome in ADAM17high expression patients. Moreover, we successfully isolated GSCs from U87glioblastoma cells via fluorescence-activated cell sorting (FACS), and demonstrated their sternness and multilineage differentiation potential in vitro. In addition, we investigated whether or not ADAM17affected the process of GSCs invasion. We found that ADAM17induced CD133+GSCs invasion via activation of the PI3K/AKT signaling pathway. These date provide direct evidence for the role of ADAM17in GSCs-mediated glioma invasion.Chapter Ⅰ ADAM17expression in gliomas of different malignancy and the correlation with patients prognosisObjective:To investigate the expression of a disintegrin and metalloprotease17(ADAM17) in gliomas of different malignancy and its correlation with patients outcome.Methods:The expression levels of ADAM17in103gliomas of different pathological grades and14temporal lobectomy of epilepsy as negative control was detected by RT-PCR, and immunohistochemical staining. The correlation between the expression levels and malignancy of the tumors were analyzed. Moreover, the correlation of expression levels of ADAM17with patients survival time was also analyzed.Results:ADAM17gene expressions were higher in glioma than that normal temporal tissue (P<0.01); Moreover, ADAM17expressions were significantly different between low-grade and high-grade gliomas. Positive correlation between the expression levels and malignancy (P<0.05) was observed. There was a highly significant difference in overall survival between ADAM17high expression and low expression patients, with poorer outcome in ADAM17high expression patients. According to the multiparametric Cox’s proportional hazard regression model analysis, ADAM17was a prognostic marker in this study.Conclusion:Detecting the expression levels of ADAM17will be helpful to evaluate the biological behaviors of gliomas and prognosis in the patients.Chapter Ⅱ Isolation, culture and identification of U87glioma stem cellsObjective:To isolate, culture and identify of glioma stem cells from human malignant glioma cell line U87MGMethods:We sorted CD133+tumor cells from human malignant glioma cell line U87MG using fluorescence-activated cell sorting (FACS). Sorted CD133+cell populations were cultured in neural basal medium with20ng/ml basic fibroblast growth factor (bFGF),20ng/ml epidermal growth factor (EGF) and100μl B27. The morphology of U87tumorspheres was observed by optic microscope, the immunophenotype and the ability of multilineage differentiation potential were tested by immunofluorescent staining and flow cytometry (FCM).Results:After sorting, U87tumorspheres were formed in serum-free medium. These tumorspheres could be digested to single-cell suspensions and formed more tumorspheres. The majority of U87tumorspheres were positive for CD133and Nestin by immunofluorescent staining and FCM. When these U87tumorspheres were transferred into serum-containing medium for differentiation, they were positive for β-tubulin Ⅲ, GFAP and Galc, showing multilineage differentiation potential.Conclusion:Glioma stem cells, which are capable of extensive proliferation, self-renewal and multi-lineage differentiation, are a small reservoir of glioma cells, existing in U87MG. Such kind of GSCs could be isolated and enriched by using fluorescence-activated cell sorting (FACS) and cultureing in serum-free neural stem cell medium.Chapter Ⅲ ADAM17promotes U87glioma stem cell migration and invasion via activation of EGFR signaling pathwayObjective:To investigated whether or not ADAM17affected the process of GSCs invasion, and expore the mechanism of EGFR signaling pathway in ADAM17-mediated GSCs migration and invasion.Methods:We examined the ADAM17expression patterns in U87GSCs by immunofluorescent staining and western-blot. The migration and invasion of U87GSCs were tested by transwell and transwell matrix penetration assays after knocking down ADAM17with shRNA and stimulation with the ADAM17agonist chemokine PMA. While phosphorylation of EGFR, AKT and ERK, western-blot. Comparisons between groups were made using unpaired Student’s t-tests, and comparisons among multiple groups were made using one-way ANOVA. Values of p<0.05were considered statistically significant.Results:GSCs expressed higher levels of ADAM17protein than non-stem tumor cells, especially when these cells formed tumorspheres in vitro. Stimulation of GSCs with the ADAM17agonist chemokine PMA increased the migrative and invasive abilities of GSCs, while knockdown of ADAM17expression in GSCs significantly decreased their migration and invasion. Inhibition of ADAM17 decreased tumor cell invasiveness through disruption of EGFR signaling pathway, and ADAM17appeared to mediate GSC invasiveness through activation of the EGFR/PI3K/AKT pathway.Conclusion:ADAM17could significantly enhance the invasive capability of GSCs through activation of the EGFR/PI3K/AKT pathway, and may provide a potential therapeutic target for glioma treatment.