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      Proteome of Glioblastoma-Derived Exosomes as a Source of Biomarkers

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          Abstract

          Extracellular vesicles (EV) are involved in important processes of glioblastoma multiforme (GBM), including malignancy and invasion. EV secreted by glioblastoma cells may cross the hematoencephalic barrier and carry molecular cargo derived from the tumor into the peripheral circulation. Therefore, the determination of the molecular composition of exosomes released by glioblastoma cells seems to be a promising approach for the development of non-invasive methods of the detection of the specific exosomal protein markers in the peripheral blood. The present study aimed to determine the common exosomal proteins presented in preparations from different cell lines and search potential glioblastoma biomarkers in exosomes. We have performed proteomics analysis of exosomes obtained from the conditioned culture medium of five glioblastoma cell lines. A list of 133 proteins common for all these samples was generated. Based on the data obtained, virtual two-dimensional electrophoresis (2DE) maps of proteins presented in exosomes of glioblastoma cells were constructed and the gene ontology (GO) analysis of exosome proteins was performed. A correlation between overexpressed in glial cell proteins and their presence in exosomes have been found. Thus, the existence of many potential glioblastoma biomarkers in exosomes was confirmed.

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          Most cited references33

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          Vimentin contributes to epithelial-mesenchymal transition cancer cell mechanics by mediating cytoskeletal organization and focal adhesion maturation

          Modulations of cytoskeletal organization and focal adhesion turnover correlate to tumorigenesis and epithelial-mesenchymal transition (EMT), the latter process accompanied by the loss of epithelial markers and the gain of mesenchymal markers (e.g., vimentin). Clinical microarray results demonstrated that increased levels of vimentin mRNA after chemotherapy correlated to a poor prognosis of breast cancer patients. We hypothesized that vimentin mediated the reorganization of cytoskeletons to maintain the mechanical integrity in EMT cancer cells. By using knockdown strategy, the results showed reduced cell proliferation, impaired wound healing, loss of directional migration, and increased large membrane extension in MDA-MB 231 cells. Vimentin depletion also induced reorganization of cytoskeletons and reduced focal adhesions, which resulted in impaired mechanical strength because of reduced cell stiffness and contractile force. In addition, overexpressing vimentin in MCF7 cells increased cell stiffness, elevated cell motility and directional migration, reoriented microtubule polarity, and increased EMT phenotypes due to the increased β1-integrin and the loss of junction protein E-cadherin. The EMT-related transcription factor slug was also mediated by vimentin. The current study demonstrated that vimentin serves as a regulator to maintain intracellular mechanical homeostasis by mediating cytoskeleton architecture and the balance of cell force generation in EMT cancer cells.
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            Mitochondria-Translocated PGK1 Functions as a Protein Kinase to Coordinate Glycolysis and the TCA Cycle in Tumorigenesis.

            It is unclear how the Warburg effect that exemplifies enhanced glycolysis in the cytosol is coordinated with suppressed mitochondrial pyruvate metabolism. We demonstrate here that hypoxia, EGFR activation, and expression of K-Ras G12V and B-Raf V600E induce mitochondrial translocation of phosphoglycerate kinase 1 (PGK1); this is mediated by ERK-dependent PGK1 S203 phosphorylation and subsequent PIN1-mediated cis-trans isomerization. Mitochondrial PGK1 acts as a protein kinase to phosphorylate pyruvate dehydrogenase kinase 1 (PDHK1) at T338, which activates PDHK1 to phosphorylate and inhibit the pyruvate dehydrogenase (PDH) complex. This reduces mitochondrial pyruvate utilization, suppresses reactive oxygen species production, increases lactate production, and promotes brain tumorigenesis. Furthermore, PGK1 S203 and PDHK1 T338 phosphorylation levels correlate with PDH S293 inactivating phosphorylation levels and poor prognosis in glioblastoma patients. This work highlights that PGK1 acts as a protein kinase in coordinating glycolysis and the tricarboxylic acid (TCA) cycle, which is instrumental in cancer metabolism and tumorigenesis.
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              Proteomic and immunologic analyses of brain tumor exosomes.

              Brain tumors are horrific diseases with almost universally fatal outcomes; new therapeutics are desperately needed and will come from improved understandings of glioma biology. Exosomes are endosomally derived 30-100 nm membranous vesicles released from many cell types into the extracellular milieu; surprisingly, exosomes are virtually unstudied in neuro-oncology. These microvesicles were used as vaccines in other tumor settings, but their immunological significance is unevaluated in brain tumors. Our purpose here is to report the initial biochemical, proteomic, and immunological studies on murine brain tumor exosomes, following known procedures to isolate exosomes. Our findings show that these vesicles have biophysical characteristics and proteomic profiles similar to exosomes from other cell types but that brain tumor exosomes have unique features (e.g., very basic isoelectric points, expressing the mutated tumor antigen EGFRvIII and the putatively immunosuppressive cytokine TGF-beta). Administration of such exosomes into syngeneic animals produced both humoral and cellular immune responses in immunized hosts capable of rejecting subsequent tumor challenges but failed to prolong survival in established orthotopic models. Control animals received saline or cell lysate vaccines and showed no antitumor responses. Exosomes and microvesicles isolated from sera of patients with brain tumors also possess EGFR, EGFRvIII, and TGF-beta. We conclude that exosomes released from brain tumor cells are biochemically/biophysically like other exosomes and have immune-modulating properties. They can escape the blood-brain barrier, with potential systemic and distal signaling and immune consequences.
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                Author and article information

                Journal
                Biomedicines
                Biomedicines
                biomedicines
                Biomedicines
                MDPI
                2227-9059
                16 July 2020
                July 2020
                : 8
                : 7
                : 216
                Affiliations
                [1 ]Orekhovich Institute of Biomedical Chemistry of Russian Academy of Medical Sciences Pogodinskaya 10, 119121 Moscow, Russia; a.t.kopylov@ 123456gmail.com (A.K.); el.petrenko@ 123456bk.ru (E.Z.)
                [2 ]Petersburg Nuclear Physics Institute NRC «Kurchatov Institute», Orlova Roshcha 1, 188300 Gatchina, Russia; voln.a@ 123456yandex.ru (A.V.); vbayramukov@ 123456gmail.com (V.B.); garaeva.luiz@ 123456yandex.ru (L.G.)
                [3 ]National Research Center “Kurchatov Institute”, Akademika Kurchatova pl. 1, 123182 Moscow, Russia; kamyshinsky.roman@ 123456gmail.com
                [4 ]Shubnikov Institute of Crystallography of Federal Scientific Research Centre ’Crystallography and Photonics” of Russian Academy of Sciences, Leninskiy Prospect 59, 119333 Moscow, Russia
                [5 ]Moscow Institute of Physics and Technology, Institutsky Lane 9, Dolgoprudny, 141700 Moscow, Russia
                [6 ]Peter the Great Saint-Petersburg Polytechnic University, Politehnicheskaya 29, 19525 St. Petersburg, Russia
                [7 ]Far Eastern Federal University, Sukhanova 8, 690091 Vladivostok, Russia; schliht@ 123456mail.ru
                Author notes
                [* ]Correspondence: naryzhnyy_sn@ 123456pnpi.nrcki.ru (S.N.); Shtam_TA@ 123456pnpi.nrcki.ru (T.S.); Tel.: +7-9111764453 (S.N.); +7-921-974-44-34 (T.S.)
                Author information
                https://orcid.org/0000-0002-7199-372X
                https://orcid.org/0000-0003-4456-6850
                https://orcid.org/0000-0002-8546-7763
                https://orcid.org/0000-0002-5702-3209
                https://orcid.org/0000-0003-0651-4785
                Article
                biomedicines-08-00216
                10.3390/biomedicines8070216
                7399833
                32708613
                3984c093-87ef-4f20-b9d5-0695fdf3c832
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 05 June 2020
                : 13 July 2020
                Categories
                Article

                glioblastoma,protein expression,proteomics,biomarkers,exosomes

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