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The Impact of Oncogenic EGFRvIII on the Proteome of Extracellular Vesicles Released from Glioblastoma Cells

2018; Elsevier BV; Volume: 17; Issue: 10 Linguagem: Inglês

10.1074/mcp.ra118.000644

1535-9484

Dongsic Choi, Laura Montermini, Dae‐Kyum Kim, Brian Meehan, Frederick P. Roth, Janusz Rak,

RNA modifications and cancer

Glioblastoma multiforme (GBM) is a highly aggressive and heterogeneous form of primary brain tumors, driven by a complex repertoire of oncogenic alterations, including the constitutively active epidermal growth factor receptor (EGFRvIII). EGFRvIII impacts both cell-intrinsic and non-cell autonomous aspects of GBM progression, including cell invasion, angiogenesis and modulation of the tumor microenvironment. This is, at least in part, attributable to the release and intercellular trafficking of extracellular vesicles (EVs), heterogeneous membrane structures containing multiple bioactive macromolecules. Here we analyzed the impact of EGFRvIII on the profile of glioma EVs using isogenic tumor cell lines, in which this oncogene exhibits a strong transforming activity. We observed that EGFRvIII expression alters the expression of EV-regulating genes (vesiculome) and EV properties, including their protein composition. Using mass spectrometry, quantitative proteomic analysis and Gene Ontology terms filters, we observed that EVs released by EGFRvIII-transformed cells were enriched for extracellular exosome and focal adhesion related proteins. Among them, we validated the association of pro-invasive proteins (CD44, BSG, CD151) with EVs of EGFRvIII expressing glioma cells, and downregulation of exosomal markers (CD81 and CD82) relative to EVs of EGFRvIII-negative cells. Nano-flow cytometry revealed that the EV output from individual glioma cell lines was highly heterogeneous, such that only a fraction of vesicles contained specific proteins (including EGFRvIII). Notably, cells expressing EGFRvIII released EVs double positive for CD44/BSG, and these proteins also colocalized in cellular filopodia. We also detected the expression of homophilic adhesion molecules and increased homologous EV uptake by EGFRvIII-positive glioma cells. These results suggest that oncogenic EGFRvIII reprograms the proteome and uptake of GBM-related EVs, a notion with considerable implications for their biological activity and properties relevant for the development of EV-based cancer biomarkers. Glioblastoma multiforme (GBM) is a highly aggressive and heterogeneous form of primary brain tumors, driven by a complex repertoire of oncogenic alterations, including the constitutively active epidermal growth factor receptor (EGFRvIII). EGFRvIII impacts both cell-intrinsic and non-cell autonomous aspects of GBM progression, including cell invasion, angiogenesis and modulation of the tumor microenvironment. This is, at least in part, attributable to the release and intercellular trafficking of extracellular vesicles (EVs), heterogeneous membrane structures containing multiple bioactive macromolecules. Here we analyzed the impact of EGFRvIII on the profile of glioma EVs using isogenic tumor cell lines, in which this oncogene exhibits a strong transforming activity. We observed that EGFRvIII expression alters the expression of EV-regulating genes (vesiculome) and EV properties, including their protein composition. Using mass spectrometry, quantitative proteomic analysis and Gene Ontology terms filters, we observed that EVs released by EGFRvIII-transformed cells were enriched for extracellular exosome and focal adhesion related proteins. Among them, we validated the association of pro-invasive proteins (CD44, BSG, CD151) with EVs of EGFRvIII expressing glioma cells, and downregulation of exosomal markers (CD81 and CD82) relative to EVs of EGFRvIII-negative cells. Nano-flow cytometry revealed that the EV output from individual glioma cell lines was highly heterogeneous, such that only a fraction of vesicles contained specific proteins (including EGFRvIII). Notably, cells expressing EGFRvIII released EVs double positive for CD44/BSG, and these proteins also colocalized in cellular filopodia. We also detected the expression of homophilic adhesion molecules and increased homologous EV uptake by EGFRvIII-positive glioma cells. These results suggest that oncogenic EGFRvIII reprograms the proteome and uptake of GBM-related EVs, a notion with considerable implications for their biological activity and properties relevant for the development of EV-based cancer biomarkers. Glioblastoma multiforme (GBM) 1The abbreviations used are:GBMglioblastoma multiformeEGFRepidermal growth factor receptorEGFRvIIIEGFR variant IIIEVsextracellular vesiclesECMextracellular matrixFBSfetal bovine serumCMconditioned mediumNTAnanoparticle tracking analysisTICtotal ion chromatogramGOgene ontologyV-SSCviolet SSC. 1The abbreviations used are:GBMglioblastoma multiformeEGFRepidermal growth factor receptorEGFRvIIIEGFR variant IIIEVsextracellular vesiclesECMextracellular matrixFBSfetal bovine serumCMconditioned mediumNTAnanoparticle tracking analysisTICtotal ion chromatogramGOgene ontologyV-SSCviolet SSC. is the most common, highly invasive astrocytic brain tumor type associated with poor prognosis, rapid cell proliferation, extensive angiogenesis, and therapeutic resistance (1Reifenberger G. Wirsching H.G. Knobbe-Thomsen C.B. Weller M. Advances in the molecular genetics of gliomas - implications for classification and therapy.Nature reviews. Clin. 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The somatic genomic landscape of glioblastoma.Cell. 2013; 155: 462-477Abstract Full Text Full Text PDF PubMed Scopus (3081) Google Scholar), demonstrable through gene transfer and targeting experiments (3Magnus N. Garnier D. Meehan B. McGraw S. Lee T.H. Caron M. Bourque G. Milsom C. Jabado N. Trasler J. Pawlinski R. Mackman N. Rak J. Tissue factor expression provokes escape from tumor dormancy and leads to genomic alterations.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 3544-3549Crossref PubMed Scopus (79) Google Scholar). glioblastoma multiforme epidermal growth factor receptor EGFR variant III extracellular vesicles extracellular matrix fetal bovine serum conditioned medium nanoparticle tracking analysis total ion chromatogram gene ontology violet SSC. glioblastoma multiforme epidermal growth factor receptor EGFR variant III extracellular vesicles extracellular matrix fetal bovine serum conditioned medium nanoparticle tracking analysis total ion chromatogram gene ontology violet SSC. EGFRvIII expression imparts aggressive and proliferative properties upon GBM cells themselves, but also acts in a non-cell-autonomous manner by deregulating pathways of intercellular communication. This includes changes in the expression of proteins involved in angiogenic, coagulant, inflammatory, immune and paracrine processes crucial for GBM progression (3Magnus N. Garnier D. Meehan B. McGraw S. Lee T.H. Caron M. Bourque G. Milsom C. Jabado N. Trasler J. Pawlinski R. Mackman N. Rak J. Tissue factor expression provokes escape from tumor dormancy and leads to genomic alterations.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 3544-3549Crossref PubMed Scopus (79) Google Scholar, 11Magnus N. Garnier D. Rak J. Oncogenic epidermal growth factor receptor up-regulates multiple elements of the tissue factor signaling pathway in human glioma cells.Blood. 2010; 116: 815-818Crossref PubMed Scopus (111) Google Scholar). Moreover, the EGFR/EGFRvIII pathway has also emerged as an important regulator of the biogenesis, intercellular trafficking and biological effects of extracellular vesicles (EVs) (12Al-Nedawi K. Meehan B. Micallef J. Lhotak V. May L. Guha A. Rak J. Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells.Nat. Cell Biol. 2008; 10: 619-624Crossref PubMed Scopus (1437) Google Scholar). EVs are heterogeneous structures ranging from 30 nm to over 1 μm in diameter and composed of a lipid bilayer that encases and protects a diverse molecular cargo (lipids, proteins and nucleic acids). 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Bourseau-Guilmain E. Bengzon J. Belting M. Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 7312-7317Crossref PubMed Scopus (678) Google Scholar, 21Fujii T. Sakata A. Nishimura S. Eto K. Nagata S. TMEM16F is required for phosphatidylserine exposure and microparticle release in activated mouse platelets.Proc. Natl. Acad. Sci. U.S.A. 2015; 112: 12800-12805Crossref PubMed Scopus (142) Google Scholar, 22Choi D.S. Choi D.Y. Hong B.S. Jang S.C. Kim D.K. Lee J. Kim Y.K. Kim K.P. Gho Y.S. Quantitative proteomics of extracellular vesicles derived from human primary and metastatic colorectal cancer cells.J. Extracell. Vesicles. 2012; 1: 1-15http://journalofextracellularvesicles.net/index.php/jev/article/view/18704Crossref Scopus (95) Google Scholar, 23Lee H.M. Choi E.J. Kim J.H. Kim T.D. Kim Y.K. Kang C. Gho Y.S. A membranous form of ICAM-1 on exosomes efficiently blocks leukocyte adhesion to activated endothelial cells.Biochem. Biophys. Res. Commun. 2010; 397: 251-256Crossref PubMed Scopus (57) Google Scholar), the causative events upstream remain largely unknown. In this regard, EGFRvIII is of interest as its expression triggers changes in the EV biogenesis, as well as their profile and biological activity (12Al-Nedawi K. Meehan B. Micallef J. Lhotak V. May L. Guha A. Rak J. Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells.Nat. Cell Biol. 2008; 10: 619-624Crossref PubMed Scopus (1437) Google Scholar). EGFRvIII oncoprotein and mRNA are found within the cargo of GBM-related EVs, an observation of considerable interest for biomarker development and given the general role of EVs as reservoirs of mutant oncoproteins in biofluids (12Al-Nedawi K. Meehan B. Micallef J. Lhotak V. May L. Guha A. Rak J. Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells.Nat. Cell Biol. 2008; 10: 619-624Crossref PubMed Scopus (1437) Google Scholar, 18Skog J. Wurdinger T. van Rijn S. Meijer D.H. Gainche L. Sena-Esteves M. Curry Jr, W.T. Carter B.S. Krichevsky A.M. Breakefield X.O. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.Nat. Cell Biol. 2008; 10: 1470-1476Crossref PubMed Scopus (3722) Google Scholar). In this context a better understanding of molecular properties associated with GBM EVs could serve as means to improve diagnostic profiling of EVs extracted from biofluids of cancer patients and to understand (and oppose) the pathogenetic potential of these vesicles (12Al-Nedawi K. Meehan B. Micallef J. Lhotak V. May L. Guha A. Rak J. Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells.Nat. Cell Biol. 2008; 10: 619-624Crossref PubMed Scopus (1437) Google Scholar). For example, whether the reported changes in the phenotype of cells that have taken up EVs from EGFRvIII expressing glioma cells are attributable to a direct influence of this oncoprotein or are related to the concomitant transfer of additional proteins remains largely unknown. In this study, we employed quantitative proteomics to analyze EVs derived from indolent parental U373 glioma cells and their EGFRvIII-expressing isogenic aggressive counterparts (U373vIII). EVs were purified using iodixanol density gradient ultracentrifugation and analyzed with UHPLC-Orbitrap Fusion Tribrid mass spectrometer. Compilation of three experimental replicates revealed remarkable EGFRvIII-related changes in the expression profiles of EV-associated proteins. Using a label-free quantitation approach, we identified a total of 1059 proteins in EV preparations from both cell lines, including 254 (24.0%) proteins significantly affected by EGFRvIII activation. This included 177 upregulated proteins in U373vIII EVs. These altered EV proteins were significantly associated with ontology terms such as focal adhesion, cell junction, cytosol, cell adhesion, and plasma membrane. Moreover, we found that U373vIII cells secrete EVs containing high levels of invasion-promoting proteins including proteases, ECM, and cell adhesion proteins. EGFRvIII transformation also caused a switch in EV tetraspanin markers (e.g. loss of CD82) (24Kowal J. Arras G. Colombo M. Jouve M. Morath J.P. Primdal-Bengtson B. Dingli F. Loew D. Tkach M. Thery C. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes.Proc. Natl. Acad. Sci. U.S.A. 2016; 113: E968-E977Crossref PubMed Scopus (1972) Google Scholar), and possibly altered some aspects of their biogenesis. Importantly, these changes were not ubiquitous but specific to individual EVs, their subpopulations and related subcellular domains. In conclusion, our results suggest that oncogenic EGFRvIII impacts the proteome of EVs released by GBM cells, and this may influence their biological activities beyond the content of EGFRvIII oncoprotein itself. All in vivo experiments were performed as described earlier (3Magnus N. Garnier D. Meehan B. McGraw S. Lee T.H. Caron M. Bourque G. Milsom C. Jabado N. Trasler J. Pawlinski R. Mackman N. Rak J. Tissue factor expression provokes escape from tumor dormancy and leads to genomic alterations.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 3544-3549Crossref PubMed Scopus (79) Google Scholar) according to the Animal Use Protocol (AUP) approved by the Institutional Animal Facility Care Committee and following Guidelines of the Canadian Council of Animal Care (CCAC). Female mice, 22–24-week-old either wild type or harboring yellow fluorescent protein transgene on the background of severe combined immunodeficiency (SCID or YFP/SCID; Charles River, Saint-Constant, QC, Canada or own colony, respectively) were inoculated subcutaneously with 5 × 106 of U373 or U373vIII cells in 0.2 ml of Matrigel HC (BD Biosciences, San Jose, CA). Tumor volume (mm3) was calculated as (width)2 × (length) × 0.5. U373 (human astrocytoma) and their EGFRvIII expressing variant (U373vIII) were described earlier (3Magnus N. Garnier D. Meehan B. McGraw S. Lee T.H. Caron M. Bourque G. Milsom C. Jabado N. Trasler J. Pawlinski R. Mackman N. Rak J. Tissue factor expression provokes escape from tumor dormancy and leads to genomic alterations.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 3544-3549Crossref PubMed Scopus (79) Google Scholar). U373vIII cells harbor Tet-off regulated EGFRvIII gene introduced by transfection, and their characteristics, generation, and maintenance were described previously (12Al-Nedawi K. Meehan B. Micallef J. Lhotak V. May L. Guha A. Rak J. Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells.Nat. Cell Biol. 2008; 10: 619-624Crossref PubMed Scopus (1437) Google Scholar). For standard culture the cells were grown in Dulbecco's modified essential medium (DMEM; Wisent, Canada) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Wisent) and 1% penicillin-streptomycin (Gibco-Life Technologies, Grand Island, NY) at 37 °C in 5% CO2. Isolation of EVs was performed as previously described (25Choi D.S. Gho Y.S. Isolation of extracellular vesicles for proteomic profiling.Methods Mol. Biol. 2015; 1295: 167-177Crossref PubMed Scopus (14) Google Scholar). Briefly, the conditioned medium (CM) was collected from cells grown for 72 h in culture media containing 10% of EV-depleted FBS (generated by centrifugation at 150,000 × g for 18 h at 4 °C). Cell viabilities were checked by trypan blue staining with three biological replicates being analyzed. Cell proliferation in 10% EV-depleted FBS media was analyzed by MTS reaction, using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) according to the manufacturer's instructions. CM was centrifuged one time at 400 × g and then passed through 0.8 μm pore-size filter. The resulting filtrate was concentrated using Amicon Ultra-15 Centrifugal Filter Unit (EMD Millipore, Billerica, MA) with 100,000 NMWL molecular cut off. The concentrate was mixed with 50% of iodixanol solution (Sigma, St. Louis, MO) and processed for density gradient ultracentrifugation at 200,000 × g for 2 h (25Choi D.S. Gho Y.S. Isolation of extracellular vesicles for proteomic profiling.Methods Mol. Biol. 2015; 1295: 167-177Crossref PubMed Scopus (14) Google Scholar). The EV-enriched fraction of iodixanol was collected (at the density of ∼1.10 g/ml) and particles were confirmed to carry CD81, and other established exosome markers (24Kowal J. Arras G. Colombo M. Jouve M. Morath J.P. Primdal-Bengtson B. Dingli F. Loew D. Tkach M. Thery C. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes.Proc. Natl. Acad. Sci. U.S.A. 2016; 113: E968-E977Crossref PubMed Scopus (1972) Google Scholar). The concentration of EV proteins was quantified using the BCA assay (Pierce Biotechnology, Rockford, IL). For concentration and size distribution of EVs nanoparticle tracking analysis (NTA) was carried out with each collected iodixanol fraction using NanoSight NS500 instrument (NanoSight Ltd., UK). Three recordings of 30 s at 37 °C were obtained and processed using NTA software (version 3.0). All experiments were carried out in three biological replicates. For proteomics, EVs were purified independently at three different times with parallel isolation on different days, each from 200 ml of conditioned media of U373 and their EGFRvIII-expressing isogenic counterpart, U373vIII. For each of the three biological replicates of LC-MS/MS, the same protein amount of EV preparation (9 μg) was desalted with SDS-PAGE, loaded onto the stacking gel followed by staining and destaining. The in-gel trypsin digestion was carried out under reducing conditions afforded by DTT, and alkylation was achieved using iodoacetic acid as previously described (26Shevchenko A. Tomas H. Havlis J. Olsen J.V. Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes.Nat. Protoc. 2006; 1: 2856-2860Crossref PubMed Scopus (3547) Google Scholar). The lyophilized peptides were resolubilized in 0.1% aqueous formic acid/2% acetonitrile, the peptides were loaded onto a Thermo Acclaim Pepmap (75 μm inner diameter × 2 cm, C18, 3 μm particle size, 100 Å pore size) (Thermo Fisher Scientific, San Jose, CA) pre-column and the onto an Acclaim Pepmap Easyspray (75 μm inner diameter × 15 cm, C18, 2 μm particle size, 100 Å pore size) (Thermo Fisher Scientific) analytical column. Separation was achieved using a Dionex Ultimate 3000 uHPLC at 220 nL/min with a gradient of 2–35% organic solvents (0.1% formic acid in acetonitrile) over 3 h. Peptides were analyzed using a Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific) operating at 120,000 resolution (FWHM in MS1, 15,000 for MS/MS) with higher-energy collisional dissociation sequencing of all peptides with a charge of 2+ or greater. The raw data were converted into *.mgf format (Mascot generic format) by MSConvert (ProteinWizard, version 3.0.6150), and searched using Mascot 2.5.1 against SwissProt (http://www.uniprot.org) human protein database (release 2016_03, 20200 entries). The tolerance was 5 ppm monoisotopic for precursor ions and 0.1 Da for fragment ions. The permission of two potential missed cleavages was selected for trypsin digestion. The following modifications were used: fixed modification for the carbamidomethylation of cysteine (58 Da) and variable modification for the oxidation of methionine (16 Da) and the deamidation of asparagine and glutamine (1 Da). The database search results were further analyzed by Scaffold Q+ software (version 4.8.4, www.proteomesoftware.com/products/scaffold/) (Proteome Sciences, Portland) (Protein threshold > 0.95%, Peptide threshold > 0.95%, and 2 of minimum number of unique peptides) (Peptide FDR: 0.8%, Protein FDR: 5.0%, Protein and peptide FDR were determined by Scaffold Q+ using the probabilistic method used by the Trans-proteomic pipeline (see http://proteome-software.wikispaces.com/FAQ-Statistics)). From three biologically replicated MS data sets of both proteomes, from U373 and U373vIII EVs, we quantified the relative protein abundance by total ion chromatogram (TIC) and calculated the p value by student's t test using Scaffold Q+ software. Proteins with less than 0.05 of p value were considered as significantly changed. The mass spectrometry proteomics data have been deposited at the ProteomeXchange Consortium via the PRIDE (27Vizcaino J.A. Csordas A. del-Toro N. Dianes J.A. Griss J. Lavidas I. Mayer G. Perez-Riverol Y. Reisinger F. Ternent T. Xu Q.W. Wang R. Hermjakob H. 2016 update of the PRIDE database and its related tools.Nucleic Acids Res. 2016; 44: D447-D456Crossref PubMed Scopus (2782) Google Scholar) partner repository with the data set identifier PXD008311 and 10.6019/PXD008311. Cell lysates and EV proteins obtained by density gradient ultracentrifugation were resolved by SDS-PAGE and then transferred to a polyvinylidene difluoride membrane. The membrane was blocked, incubated with primary antibody followed by the secondary antibody conjugated with horseradish peroxidase, and subjected to the enhanced chemiluminescence. All images were acquired by a ChemiDoc MP imager (Bio-Rad, Hercules, CA). Rabbit anti-CD82, rabbit anti-EGFR, rabbit anti-ITGA6, rabbit anti-ITGB4, and goat anti-rabbit antibodies were purchased from Cell Signaling Technology (Beverly, MA). Rabbit anti-CANX, rabbit anti-CD44, mouse anti-CD81, rabbit anti-CD9, rabbit anti-GAPDH, rabbit anti-SDCB1 antibodies were purchased from Abcam (Cambridge, MA). Mouse anti-actin and goat anti-mouse IgG were purchased from Sigma. The lists of identified proteins were imported into the DAVID Bioinformatics database (http://david.abcc.ncifcrf.gov) and assigned to their GO annotations (cellular component and biologi