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Quantitative proteomic analysis of large and small extracellular vesicles from prostate cancer cells

ABSTRACT: Extracellular vesicles (EVs) are membranous structures of variable size, shed from diverse cell types including highly invasive, fast migrating, amoeboid tumor cells. Silencing of the Diaphanous-related formin-3 (DIAPH3) protein in DU145 prostate cancer cells induces an amoeboid phenotype characterized by membrane blebbing and shedding of an atypically large population of EVs termed large oncosomes. We employed this cell system to characterize, for the first time, the global protein composition of large oncosomes and small EVs. The salient finding of this study, revealed by SILAC-based LC-MS/MS analysis was the identification of 20 unique proteins in large oncosomes and 7 in small EVs. Additionally, both types of EV were significantly enriched in proteins with cancer-related functions, suggesting that tumor interactions with the microenvironment are influenced by EV cargo. Proteins belonging to a series of functional classes, including GTPases, ESCRTs, RNA-binding proteins and membrane proteins, often identified in and which are sometimes considered specific for exosomes, appeared in both EV populations. Proteins such as CK18 and FN1, significantly enriched in large oncosomes, were selected to develop an assay to detect large oncosomes in the circulation. Finally, proteins over-represented in large oncosomes versus small EVs with at least a four-fold enrichment were significantly associated with cell migration, docetaxel resistance, angiogenesis, and prostate cancer bone metastasis, suggesting a distinct functional role in tumor progression for this larger class of EV. These observations indicate that, despite their origination from the same cell donors, the two distinct EV populations are enriched in different proteins and contribute diversified functions. These findings underscore the need for additional in-depth analyses to understand the unique contribution of subpopulations of EVs to tumor progression.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Prostate Adenocarcinoma

SUBMITTER: Wei Yang

LAB HEAD: Wei Yang

PROVIDER: PXD000776 | Pride | 2015-03-18

REPOSITORIES: Pride

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Project description:Circulating extracellular vesicles (EVs) contain molecular footprints - lipids, proteins, metabolites, RNA, and DNA - from their cell of origin and may provide non-invasive access for detection, characterization, and monitoring of diseases. Consequently, EV-associated RNA and proteins have gained widespread interest in developing liquid-biopsy assays for cancer. Yet, an integrative proteo-transcriptomic landscape of EVs and a direct comparison with their cell of origin remains relatively unexplored. Here, we report that EVs enrich for distinct molecular cargo and their proteo-transcriptome does not linearly correlate with their cell of origin. Our sub-cellular compartment analyses revealed that EV enrich 2-6 times more cytoskeletal and extracellular proteins, while their donor cells cargo 3-10 times more nuclear and mitochondrial proteins. EVs predominantly enrich (40-60%) for small RNA between ~15-200 nucleotides, which include miRNAs, siRNAs, tRNA, 5S rRNAs, and snRNAs, associated with cell differentiation, development, and Wnt signaling signatures. Finally, our integrated proteo-transcriptomic analyses reveal that EVs are enriched with specific small RNAs (RNY3, vtRNA, and MIRLET-7) and their complementary proteins (YBX1, IGF2BP2, SRSF1/2). These analyses suggest that RNA-protein complexes may constitute a functional interaction network inside EVs to protect and regulate access to EV-RNA. To ensure that our analyses are unbiased and independent of the cell of origin, EV isolation methods, site of experimentation, site of data generation, and methodologies applied for RNAseq and mass spectrometry, we have generated and curated comprehensive datasets of proteomics and transcriptomics from a total of 12 cancer cell lines, their EVs, and prostate cancer patientsÕ serum EVs (A detailed description is provided in Fig. 1). These combined analyses demonstrated that irrespective of the cell of origin and other aforementioned conditions, EVs enrich for distinct RNA and protein signatures that do not linearly correlate with their cell of origin.

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Quantitative proteomic analysis of large and small extracellular vesicles from prostate cancer cells

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