Endosomal trafficking of the EGFR mutant, EGFRvIII, results in exosomal secretion that triggers astrocyte reactivity

Abstract Background: The epidermal growth factor receptor (EGFR) variant three (EGFRvIII) mutation is linked with approximately one third of Glioblastoma Multiforme (GBM) tumors and is associated with poor patient prognosis. Persistent signaling due to a lack of the EGFR ectodomain and inefficient degradation have been suggested to underlie the tumorgenic properties of EGFRvIII. Methods: Cell viability and trans-well migration assays were used to determine the effects that expression of the oncoprotein, EGFRvIII, had on glioma cells. A cell-free reconstitution assay developed by our laboratory was utilized to determine trafficking of EGFR and EGFRvIII at the late endosome and determine molecular requirements for inward budding of proteins into the MVB. Western Blot Analysis and Nanosight Tracking Analysis were used to characterize exosomes by protein marker presence and vesicle size, respectively. Immunohistochemistry and Western Blot analysis were used to determine astrocyte reactivity marked by GFAP expression. Results: Like the parental EGFR, we observed that EGFRvIII is internalized into the intraluminal vesicles of late endosomes / multivesicular bodies (MVBs) but does not follow the canonical pathway by which wild-type EGFR is degraded following MVB fusion with lysosomes. These studies suggested that EGFRvIII is secreted on exosomes, the intraluminal vesicles that are secreted upon MVB fusion with the plasma membrane, suggested that EGFRvIII is localized in a subset of MVBs that preferentially fuse with the plasma membrane rather than with lysosomes which may account for its decreased degradation. Astrocytes are a component of the GBM tumor microenvironment with which tumor cells interact in a paracrine manner. EGFRvIII-containing exosomes derived from GBM cells induce changes in astrocytes that mimic reactive astrogliosis including an increase in glial fibrillary acidic protein (GFAP). Conclusions: These studies reveal novel aspects of the endocytic trafficking of EGFRvIII that underlie its reduced degradation and the mechanism by which it is packaged into exosomes for secretion. Moreover, EGFRvIII secretion on exosomes can facilitate changes in the tumor microenvironment to enable tumor growth.