Abstract
The aim of this study was to identify the receptor for glial cell line-derived neurotrophic factor (GDNF) in glioblastoma multiforme (GBM). After GST pull-down assays, membrane proteins purified from C6 rat glioma cells were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). The differentially expressed proteins were annotated using Gene Ontology, and neuropilin-1 (NRP1) was identified as the putative GDNF receptor in glioma. NRP1 was more highly expressed in human GBM brains and C6 rat glioma cells than in normal human brains or primary rat astrocytes. Immunofluorescence staining showed that NRP1 was recruited to the membrane by GDNF, and NRP1 co-immunoprecipitated with GDNF. Using the NRP1 and GDNF protein structures to assess molecular docking in the ZDOCK server and visualization with the PyMOL Molecular Graphics System revealed 8 H-bonds and stable positive and negative electrostatic interactions between NRP1 and GDNF. RNAi knockdown of NRP1 reduced proliferation of C6 glioma cells when stimulated with GDNF. NRP1 was an independent risk factor for both survival and recurrence in GBM patients. High NRP1 mRNA expression correlated with shorter OS and DFS (OS: χ2=4.6720, P=0.0307; DFS: χ2=11.013, P=0.0009). NRP1 is thus a GDNF receptor in glioma cells and a potential therapeutic target.
Highlights
Glioblastoma (GBM) is one of the most common primary tumors of the central nervous system (CNS) with poor survival outcomes
Since glial cell line-derived neurotrophic factor (GDNF) is secreted in high amounts by gliomas, we postulated that GDNF membrane receptors would be highly expressed in gliomas
We compared the expression of GDNF family receptor alpha 1 (GFRA1), receptor tyrosine kinase (RET), NCAM1, Integrin beta-1 (ITGB1), CDH2 and SDC3 between glioblastoma multiforme (GBM) and normal brains in the TCGA database using the Oncomine® Platform
Summary
Glioblastoma (GBM) is one of the most common primary tumors of the central nervous system (CNS) with poor survival outcomes. It is characterized by uncontrolled cellular proliferation that is driven by a complex integration of a various extracellular stimuli that signal through multiple membrane receptor systems, resulting in elaborate changes in gene expression [1]. Glial cell line-derived neurotrophic factor (GDNF), a member of the GDNF family of ligands (GFL), is strongly expressed in human gliomas [3]. Knock-down of GDNF and its binding receptor, GDNF family receptor alpha 1 (GFRA1) decreases the proliferation of C6 glioma cells [4]. The mechanisms downstream of GDNF binding to C6 cells are not clear because of multiple signal transducing receptors for GDNF in different cells
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