Oncogenic H-Ras reprograms Madin-Darby canine kidney (MDCK) cell-derived exosomal proteins following epithelial-mesenchymal transition
journal contributionposted on 12.08.2021, 06:32 authored by BJ Tauro, RA Mathias, David GreeningDavid Greening, SK Gopal, H Ji, EA Kapp, BM Coleman, Andrew HillAndrew Hill, U Kusebauch, JL Hallows, D Shteynberg, RL Moritz, HJ Zhu, Richard SimpsonRichard Simpson
Epithelial-mesenchymal transition (EMT) is a highly conserved morphogenic process defined by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype. EMT is associated with increased aggressiveness, invasiveness, and metastatic potential in carcinoma cells. To assess the contribution of extracellular vesicles following EMT, we conducted a proteomic analysis of exosomes released from Madin-Darby canine kidney (MDCK) cells, and MDCK cells transformed with oncogenic H-Ras (21D1 cells). Exosomes are 40-100 nm membranous vesicles originating from the inward budding of late endosomes and multivesicular bodies and are released from cells on fusion of multivesicular bodies with the plasma membrane. Exosomes from MDCK cells (MDCK-Exos) and 21D1 cells (21D1-Exos) were purified from cell culture media using density gradient centrifugation (OptiPrep™), and protein content identified by GeLCMS/ MS proteomic profiling. Both MDCK- and 21D1-Exos populations were morphologically similar by cryo-electron microscopy and contained stereotypical exosome marker proteins such as TSG101, Alix, and CD63. In this study we show that the expression levels of typical EMT hallmark proteins seen in whole cells correlate with those observed in MDCK- and 21D1-Exos, i.e. reduction of characteristic inhibitor of angiogenesis, thrombospondin-1, and epithelial markers E-cadherin, and EpCAM, with a concomitant up-regulation of mesenchymal makers such as vimentin. Further, we reveal that 21D1-Exos are enriched with several proteases (e.g. MMP-1, -14, -19, ADAM-10, and ADAMTS1), and integrins (e.g. ITGB1, ITGA3, and ITGA6) that have been recently implicated in regulating the tumor microenvironment to promote metastatic progression. A salient finding of this study was the unique presence of key transcriptional regulators (e.g. the master transcriptional regulator YBX1) and core splicing complex components (e.g. SF3B1, SF3B3, and SFRS1) in mesenchymal 21D1-Exos. Taken together, our findings reveal that exosomes from Ras-transformed MDCK cells are reprogrammed with factors which may be capable of inducing EMT in recipient cells. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
This work was supported by the National Health & Medical Research Council (NHMRC) of Australia for program grant #487922 (RJS, JH, DWG), grants #280913 and #433619 (H-JZ), grants #628946 and #400202 (AFH). AFH is also supported by an Australian Research Council (www.arc.gov.au) Future Fellowship (FT100100560). RAM is supported by an Early Career CJ Martin Fellowship #APP1037043, and BMC by an NHMRC Dora Lush Biomedical Postgraduate Scholarship #628959. BJT is supported by The University of Melbourne Research Scholarship. Analysis of proteomic data described in this work was supported using the Australian Proteomics Computational Facility funded by the National Health & Medical Research Council of Australia grant #381413. Electron microscopy was performed at the Advanced Microscopy Facility at the Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne. This work was also supported, in part, by American Recovery and Reinvestment Act funds through National Institutes of Health Grant R01 HG005805 (RLM), the NIGMS Grant 2P50 GM076547 from Center for Systems Biology, the Luxembourg Centre for Systems Biomedicine and the University of Luxembourg, and from the National Science Foundation (MRI Grant 0923536). UK was supported by a fellowship from the German Academic Exchange Service. We thank the NCI of the NIH for support (Grant #1R03CA156667 to RLM).
JournalMolecular and Cellular Proteomics
PublisherAMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
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Science & TechnologyLife Sciences & BiomedicineBiochemical Research MethodsBiochemistry & Molecular BiologyBREAST-CANCERMATRIX METALLOPROTEINASE-1METASTATIC NICHETYROSINE KINASETUMOR-GROWTHMICROVESICLESINVASIONYB-1ANGIOGENESISEXPRESSIONAnimalsDogsCell Transformation, Neoplasticras ProteinsPeptide HydrolasesAnnexinsIntegrinsProteomeGenes, rasExosomesEpithelial-Mesenchymal TransitionTetraspaninsMadin Darby Canine Kidney Cells