Abstract LB-514: Contribution of ADAM17 to Kit ligand ectodomain shedding in high-grade gliomas

Abstract

Abstract Background Malignant anaplastic astrocytoma and glioblastoma are considered to derive from astrocytic precursor cells. They display high vascularization, aggressive growth and invasion into the surrounding brain tissue, and respond poorly to conventional treatments. A disintegrin and metalloproteinases (ADAMs) are membrane-associated metalloproteinases with a complex multi-domain structure. Kit ligand (Kitl), also referred to as stem cell factor (SCF) is synthesized as a membrane-anchored precursor that can be processed to produce the soluble growth factor. As a ligand for Kit receptor tyrosine kinase, Kitl plays important roles in activation of various signalling pathways leading to cell proliferation, differentiation, migration and survival. Previous data has shown that ADAM17 affects Kitl shedding. Hypoxia-induced ADAM17 has been reported to contribute to glioma cell invasion whereas Kitl expression has been demonstrated in glioma cell lines but its function remains unclear. As invasive behaviour is a hallmark of high-grade gliomas, the main aim of our recent study is to investigate the functional role and contribution of ADAM17 to ectodomain shedding of Kitl in these tumors. Methods Quantitative RT-PCR analyses for ADAM17 and Kitl were performed using 36 adult glioma tumor samples including 9 pilocytic astrocytomas WHO grade I (PA I), 9 diffuse astrocytomas WHO grade II (DA II), 9 anaplastic astrocytomas WHO grade III (AA III) and 9 glioblastoma multiforme WHO grade IV (GBM) as well as three (LN-229, LN-308 and U87-MG) glioma cell lines. ADAM17 endogenous expression in glioma cell lines was knocked-down using ADAM17-specific short hairpin RNA (shRNA). The phenotype of the stable transfectants was analyzed by proliferation and migration assays. Immunoblotting and ELISA were performed to detect the Kitl ectodomain released in the cell supernatants. Results We found that ADAM17 and KITLG are overexpressed in high-grade gliomas when compared with low-grade gliomas and normal brain tissue and the higher mRNA levels correlated with the tumor grade. Knock-down of ADAM17 by shRNA significantly reduced glioma cell migration. Moreover shedding of Kitl from control cells (scrambled transfected cells) can be stimulated by addition of phorbol 12-myristate 13-acetate (PMA) together with Ionomycin for 1 hour. In contrast, the PMA/Ionomycin-stimulated Kitl shedding was completely prevented in ADAM17-knock-down cells indicating that ADAM17 is an important PMA/Ionomycin -responsive Kitl sheddase in glioma cells. These results provided evidence for an involvement of ADAM17 in PMA/Ionomycin stimulation of Kitl shedding in glioma cells. Conclusion Our results suggest that ADAM17 in high-grade gliomas effects Kitl ectodomain shedding and may substantially contribute to glioma cell migration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-514. doi:1538-7445.AM2012-LB-514