Secreted Glioblastoma Nanovesicles Contain Intracellular Signaling Proteins and Active Ras Incorporated in a Farnesylation-dependent Manner

Abstract

Edited by Alex TokerGlioblastomas (GBMs) are malignant brain tumors with a median survival of less than 18 months. Redundancy of signaling pathways represented within GBMs contributes to their therapeutic resistance. Exosomes are extracellular nanovesicles released from cells and present in human biofluids that represent a possible biomarker of tumor signaling state that could aid in personalized treatment. Herein, we demonstrate that mouse GBM cell-derived extracellular nanovesicles resembling exosomes from an H-RasV12 myr-Akt mouse model for GBM are enriched for intracellular signaling cascade proteins (GO: 0007242) and Ras protein signal transduction (GO: 0007265), and contain active Ras. Active Ras isolated from human and mouse GBM extracellular nanovesicles lysates using the Rasbinding domain of Raf also coprecipitates with ESCRT (endosomal sorting complex required for transport)-associated exosome proteins Vps4a and Alix. Although we initially hypothesized a role for active Ras protein signaling in exosome biogenesis, we found that GTP binding of K-Ras was dispensable for its packaging within extracellular nanovesicles and for the release of Alix. By contrast, farnesylation of K-Ras was required for its packaging within extracellular nanovesicles, yet expressing a K-Ras farnesylation mutant did not decrease the number of nanovesicles or the amount of Alix protein released per cell. Overall, these results emphasize the primary importance of membrane association in packaging of extracellular nanovesicle factors and indicate that screening nanovesicles within human fluids could provide insight into tissue origin and the wiring of signaling proteins at membranes to predict onset and behavior of cancer and other diseases linked to deregulated membrane signaling states.

Ras-AKT signaling represses the phosphorylation of histone H1.5 at threonine 10 via GSK3 to promote the progression of glioma

“…Numerous evidences suggest that the occurrence of glioma is related with the activation of multiple oncogenes, including Ras family genes [3]. The family of Ras contains three members: K-Ras, H-Ras, and N-Ras.…”

Failure to detect functional transfer of active K-Ras protein from extracellular vesicles into recipient cells in culture

“…In a previous publication, we demonstrated that GBM exosomes are enriched for signaling proteins and contain active GTP-bound Ras, and that active Ras interacts with exosome proteins based on GST-Raf-RBD pulldown data [ 1 ]. In light of these results, we wondered whether GBM exosomes are taken up by other GBM cells to influence their signaling status.…”

“…Exosomes are tiny (50-150nm) extracellular vesicles (EVs) implicated in cell-to-cell communication. When compared to intact cells, these vesicles are enriched for membrane-associated signaling and cell communication proteins [ 1 ], and have been proposed to alter a number of cellular processes, such as prion protein transmission and neurodegenerative diseases [ 2 ], regulation of immune functions [ 3 ], tumor angiogenesis [ 4 – 16 ], fibroblast signaling to tumors [ 17 , 18 ], and priming of the metastatic niche [ 19 – 26 ]. In addition to lipids and proteins, these vesicles carry DNA and RNA [ 13 , 27 – 29 ] and are present in biofluids; hence, research is underway to harness these carriers of cell communication cargoes to identify signatures that could be useful biomarkers in human disease [ 30 – 35 ].…”

“…We initially hypothesized that active Ras might be transferred to other cells and could allow a heterogeneous population of cells to directly share membrane signaling factors and respond more effectively to stress. In a previous study, we found that glioblastoma (GBM) brain tumor cells release active Ras in extracellular nanovesicles whose properties are consistent with exosomes [ 1 ]. In this work, we observed that lipophilic dye-labeled exosomes from GBM cells transferred dye to the cytoplasm of GBM cells and found that treating serum-starved cells with these exosomes exogenously improved their growth over time.…”

Proteomics of the Salivary Fluid

“…Proteomics is a technology enabling significantly and profoundly better approach to investigating and understanding proteins' function and their posttranslational modifications [7][8][9][10][11][12]. Applying proteomics methods for analysis of clinical samples is especially important in time of personalized medicine, which tailors individualized treatment of each patient based on specification of the diseases [3,[13][14][15][16][17][18][19]. Furthermore, (clinical) proteomics can be used as a method-of-choice for the screening of biomarkers used for early discovery and early diagnostics.…”