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Berlin Brandenburg

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  • 1
    Language: English
    In: IUBMB life, March 2019, Vol.71(3), pp.364-375
    Description: Emerging evidence on efficient tumor growth regulation by endogenous lectins directs interest to determine on a proof-of-principle level the range of information on alterations provided by full-scale analysis using phosphoproteomics. In our pilot study, we tested galectin-4 (gal-4) that is a growth inhibitor for colon cancer cells (CRC), here working with the LS 180 line. In order to cover monitoring of short- and long-term effects stable isotope labeling by amino acids in cell culture-based quantitative phosphoproteomic analyses were conducted on LS 180 cell preparations collected 1 and 72 h after adding gal-4 to the culture medium. After short-term treatment, 981 phosphosites, all of them S/T based, were detected by phosphoproteomics. Changes higher than 1.5-fold were seen for eight sites in seven proteins. Most affected were the BET1 homolog (BET1), whose level of phosphorylation at S50 was about threefold reduced, and centromere protein F (CENPF), extent of phosphorylation at S3119 doubling in gal-4-treated cells. Phosphoproteome analysis after 72 h of treatment revealed marked changes at 33 S/T-based phosphosites from 29 proteins. Prominent increase of phosphorylation was observed for cofilin-1 at position S3. Extent of phosphorylation of the glutamine transporter SLC1A5 at position S503 was decreased by a factor of 3. Altered phosphorylation of BET1, CENPF, and cofilin-1 as well as a significant effect of gal-4 treatment on glutamine uptake by cells were substantiated by independent methods in the Vaco 432, Colo 205, CX 1, and HCT 116 cell lines. With the example of gal-4 which functions as a tumor suppressor in CRC cells, we were able to prove that cell surface binding of the lectin not only markedly influences the cell proteome, but also has a bearing on malignancy-associated intracellular protein phosphorylation. These results underscore the potential of this approach to give further work on elucidating the details of signaling underlying galectin-triggered growth inhibition a clear direction. © 2018 IUBMB Life, 71(3):364-375, 2019.
    Keywords: Glutamine Transporter ; Colorectal Cancer ; Galectin-4 ; Growth-Inhibition ; Phosphoproteome ; Tumor Suppressor
    ISSN: 15216543
    E-ISSN: 1521-6551
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  • 2
    Language: English
    In: Journal of proteome research, 02 December 2016, Vol.15(12), pp.4412-4422
    Description: Endogenous lectins have the capacity to translate glycan-encoded information on the cell surface into effects on cell growth. As test cases to examine changes in protein presence associated with tumor growth inhibition, we applied SILAC-based proteomics on human colon carcinoma cells treated with galectin-4 (Gal-4). The five tested lines-LS 180, Vaco 432, Colo 205, CX 1, and HCT 116-responded with differentiation and reduced proliferation to Gal-4 binding. In proteomic analysis (mass spectral data deposited with PRIDE, PXD003489), 2654 proteins were quantified, of which 190 were down-regulated and 115 were up-regulated (〉2-fold). 1D annotation analysis of the results indicated down-regulation of DNA replication-associated processes, while protein presence for secretory and transport functions appeared increased. The strongest induction was found for CALB2 (calretinin; ∼24-fold), TGM2 (protein-glutamine γ-glutamyltransferase 2; ∼11-fold), S100A3 (∼10-fold), and GSN (gelsolin; 9.5-fold), and the most pronounced decreases were seen for CDKN2A (tumor suppressor ARF; ∼6-fold), EPCAM (epithelial cell adhesion molecule; ∼6-fold), UBE2C (ubiquitin-conjugating enzyme E2 C; ∼5-fold), KIF2C (kinesin-like protein KIF2C; 5-fold), and LMNB1 (lamin-B1; ∼5-fold). The presence of the common proliferation marker Ki-67 was diminished about 4-fold. By tracing significant alterations of protein expression likely relevant for the observed phenotypic effects, the capacity of a galectin to affect the proteome of human colon cancer cells at multiple sites is revealed.
    Keywords: Silac ; Adhesion ; Cancer ; Lectin ; Malignancy ; Proliferation ; Cell Membrane -- Metabolism ; Colonic Neoplasms -- Metabolism ; Galectin 4 -- Pharmacology ; Proteome -- Drug Effects
    ISSN: 15353893
    E-ISSN: 1535-3907
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  • 3
    Language: English
    In: Cell Communication and Signaling, 01 April 2017, Vol.15(1), pp.1-14
    Description: Abstract Background Colorectal cancers (CRCs) that lack DNA mismatch repair function exhibit the microsatellite unstable (MSI) phenotype and are characterized by the accumulation of frameshift mutations at short repetitive DNA sequences (microsatellites). These tumors recurrently show inactivating...
    Keywords: Exosomes ; Intercellular Communication ; Proteomics ; Transforming Growth Factor Beta Receptor Type 2 ; DNA Mismatch Repair Deficiency ; Microsatellite Instability ; Biology
    E-ISSN: 1478-811X
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  • 4
    Language: English
    In: TGFBR2-dependent alterations of exosomal cargo and functions in DNA mismatch repair-deficient HCT116 colorectal cancer cells. Cell Communication and Signaling, 15(1), 14., United Kingdom. (2017).
    Description: BACKGROUND: Colorectal cancers (CRCs) that lack DNA mismatch repair function exhibit the microsatellite unstable (MSI) phenotype and are characterized by the accumulation of frameshift mutations at short repetitive DNA sequences (microsatellites). These tumors recurrently show inactivating frameshift mutations in the tumor suppressor Transforming Growth Factor Beta Receptor Type 2 (TGFBR2) thereby abrogating downstream signaling. How altered TGFBR2 signaling affects exosome-mediated communication between MSI tumor cells and their environment has not been resolved. Here, we report on molecular alterations of exosomes shed by MSI cells and the biological response evoked in recipient cells. METHODS: Exosomes were isolated and characterized by electron microscopy, nanoparticle tracking, and western blot analysis. TGFBR2-dependent effects on the cargo and functions of exosomes were studied in a MSI CRC model cell line enabling reconstituted and inducible TGFBR2 expression and signaling. Microsatellite frameshift mutations in exosomal and cellular DNA were examined by PCR-based DNA fragment analysis and exosomal protein profiles were identified by mass spectrometry. Uptake of fluorescent-labeled exosomes by hepatoma recipient cells was monitored by confocal microscopy. TGFBR2-dependent exosomal effects on secreted cytokine levels of recipient cells were analyzed by Luminex technology and ELISA. RESULTS: Frameshift mutation patterns in microsatellite stretches of TGFBR2 and other MSI target genes were found to be reflected in the cargo of MSI CRC-derived exosomes. At the proteome level, reconstituted TGFBR2 expression and signaling uncovered two protein subsets exclusively occurring in exosomes derived from TGFBR2-deficient (14 proteins) or TGFBR2-proficient (five proteins) MSI donor cells. Uptake of these exosomes by recipient cells caused increased secretion (2-6 fold) of specific cytokines (Interleukin-4, Stem Cell Factor, Platelet-derived Growth Factor-B), depending on the TGFBR2 expression status of the tumor cell. CONCLUSION: Our results indicate that the coding MSI phenotype of DNA mismatch repair-deficient CRC cells is maintained in their exosomal DNA. Moreover, we uncovered that a recurrent MSI tumor driver mutation like TGFBR2 can reprogram the protein content of MSI cell-derived exosomes and in turn modulate the cytokine secretion profile of recipient cells. Apart from its diagnostic potential, these TGFBR2-dependent exosomal molecular and proteomic signatures might help to understand the signaling routes used by MSI tumors. Fricke et al. uncovered coding microsatellite instability-associated mutations of colorectal tumor driver genes like TGFBR2 in MSI tumor cellderived exosomes. Depending on the TGFBR2 expression status of their donor cells, shed exosomes show distinct proteomic signatures and promote altered cytokine secretion profiles in recipient cells.
    Description: Peer reviewed
    Keywords: Chemokines/Metabolism ; Colorectal Neoplasms/Metabolism ; Dna Mismatch Repair ; Enzyme-Linked Immunosorbent Assay ; Exosomes/Metabolism/Ultrastructure ; Frameshift Mutation/Genetics ; Hct116 Cells ; Hep G2 Cells ; Humans ; Microsatellite Instability ; Platelet-Derived Growth Factor/Metabolism ; Protein-Serine-Threonine Kinases/Metabolism ; Proteome/Metabolism ; Receptor, Transforming Growth Factor-Beta Type Ii ; Receptors, Transforming Growth Factor Beta/Metabolism ; Reproducibility Of Results ; Colorectal Cancer ; Dna Mismatch Repair Deficiency ; Exosomes ; Intercellular Communication ; Microsatellite Instability ; Proteomics ; Transforming Growth Factor Beta Receptor Type 2 ; Human Health Sciences :: Oncology ; Sciences De La Santé Humaine :: Oncologie
    Source: ORBi (Open Repository and Bibliography), University of Liège
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  • 5
    Language: English
    In: International Journal of Molecular Sciences, 01 August 2019, Vol.20(17), p.4162
    Description: Microsatellite unstable (MSI) colorectal cancers (CRCs) are characterized by mutational inactivation of Transforming Growth Factor Beta Receptor Type 2 (TGFBR2). TGFBR2-deficient CRCs present altered target gene and protein expression. Such cellular alterations modulate the content of CRC-derived...
    Keywords: Extracellular Vesicles ; Exosomes ; Proteomics ; Stable Isotope Labeling With Amino Acids in Cell Culture (Silac) ; Tgfbr2 ; Microsatellite Instability ; DNA Mismatch Repair ; Colorectal Cancer ; Biology
    ISSN: 16616596
    E-ISSN: 1422-0067
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