A Positive Feedback Loop Between ADAM12 And Notch Pathway Maintains Self-renewal Of CD133+ Glioma Cells

Malignant glioma is the most common primary tumor with highly invasive biological characteristic in the central nervous system. The may be the key reason for local recurrence of glioma after radical tumor resection and adjuvant modalities such as radiotherapy and chemotherapy. Recent studies found that glioma stem cells(GSCs) might play an key role in tumorigenesis and development of malignant gliomas. The activation of Notch pathway maintains the stemness phenotype of neural stem cells via regulation of target genes, such as nestin, etc. The same role of Notch pathway in GSCs also has been demonstrated.The stemness phenotype of Glioma stem cells needs activation of Notch pathway, and glioma stem cells present overexpression of Notch receptors and low expression of Delta-like ligands because of characteristic of Notch pathway. However, how GSCs to maintain this state in malignant glioma? A disintegrin and metalloprotease12(ADAM12) is a multifunctional protein belonging to the ADAMs family, which can proteolysis other transmembrane proteins in the same cell, such as DLL1. Aimed at finding effective drug targets, we explored the crosstalk between ADAM12 and Notch pathway on self-renewal capacity in GSCs. The study included three parts:1.The expression of ADAM12 in different pathological grades of glioma tissues.Different grades of glioma tissues and normal brain tissues were selected to be detected expression and distribution of ADAM12. Then, we detected expression of ADAM12 in specimens at level of mRNA and protein. Expression of ADAM12, CD133, nestin and Notch1 were tested in glioblastoma specimens. ADAM12 and stem cell markers were tested and verified the co-expression in specimens and neurosphere. In the immunohistochemistry analysis, the results revealed that the expression of ADAM12 was negative or just weakly positive in normal brain tissues, but strongly expressed in glioma tissues and the expression level was positively correlated with the pathological grades of glioma tissues. The results of western blotting and Real time-PCR were consistent with immunohistochemistry results. ADAM12, CD133, nestin and Notch1 presented overexpression in glioblastoma specimen, furthermore they presented tendency of co-expression in immunofluorescence staining.2.Effect of ADAM12 silenced by shRNA on self-renewal capacity of CD133 positive glioma cells.Firstly, CD133 positive glioma cells were selected from U87 and U251 glioma cell lines by immunomagnetic beads, which would be cultured in serum-free medium. The shRNA recombinant lentivirus aimed at silencing ADAM12 was prepared. CD133 positive glioma cells were employed in this study and assigned into three groups: group of ADAM12 silenced by shRNA1(shRNA1-ADAM12), group of ADAM12 silenced by shRNA2(shRNA2-ADAM12) and non-silencing ADAM12 group(shRNA-C). Based on the established stably transfected cell lines, the following experiments were performed: ADAM12 expression was detected at mRNA and protein level respectively. Self-renewal was tested by tumor sphere formation assay, and molecular markers were detected at protein level. The expression of Notch1, DLL1 and Hes1 were detected at protein level and mRNA level respectively. ELISA assay was adopted to testing shedding DLL1 in cell medium. The results showed that the mRNA and protein expression levels of ADAM12 were significantly down-regulated after transfecting ADAM12 shRNA. Tumor sphere formation ability of shRNA1- ADAM12 and shRNA2-ADAM12 groups were lower than that of shRNA-C group. The protein expression of GFAP and TUBB3 were up-regulated, whereas the protein expression levels of CD133, nestin and Hes1 were down-regulated in treated groups. Expression of Hes1 was down-regulated at either mRNA or protein level; expression of Notch1 had no significant difference at either protein or mRNA level; expression of DLL1 at protein level in treated groups were significantly up-regulated, whereas expression of DLL1 at mRNA level had no significant difference; shedding DLL1 in medium increased significantly. The result of U87 cell lines was consistent with that of U251 cell lines.3.Investigating the expression of ADAM12 regulated by Notch pathway in CD133 positive glioma cells.The specific inhibitor DAPT aimed at inhibiting Notch pathway and the shRNA recombinant lentivirus aimed at silencing Notch1 were prepared. CD133 positive glioma cells were employed and assigned into three groups: DAPT group, group of Notch1 silenced by shRNA(shRNA-Notch1) and non-treatment group(control). Compared with control group, the expression of ADAM12 in DAPT group and shRNA-Notch1 group were significantly down-regulated at protein and mRNA level respectively. The result of U87 cell lines was consistent with that of U251 cell lines. The conclusions of this study were drawn as follows:1. Expression of ADAM12 in glioma tissues remarkably higher than that in normal brain tissue, and the expression level presented positive correlation with WHO grade of glioma.2. Silencing of ADAM12 gene could inhibit self-renewal capacity of CD133 positive glioma cells.3. DLL1 ectodomain was cleaved by ADAM12 hydrolysis, which would promote Notch pathway activity via removing cis-inhibition.4. Activation of the Notch pathway can promote the ADAM12 gene expression in the CD133 positive glioma cells5. The positive feedback loop of ADAM12-Notch pathway maintain self-renewal of CD133-positive glioma cells.6. ADAM12 is expected to be a drug target against glioma stem cell.