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  • Gebert, Johannes  (7)
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  • 1
    Language: English
    In: PLoS ONE, 01 January 2015, Vol.10(6), p.e0131506
    Description: Although inactivating frameshift mutations in the Transforming growth factor beta receptor type 2 (TGFBR2) gene are considered as drivers of microsatellite unstable (MSI) colorectal tumorigenesis, consequential alterations of the downstream target proteome are not resolved completely. Applying a click-it chemistry protein labeling approach combined with mass spectrometry in a MSI colorectal cancer model cell line, we identified 21 de novo synthesized proteins differentially expressed upon reconstituted TGFBR2 expression. One candidate gene, the TGF-ß family member Growth differentiation factor-15 (GDF-15), exhibited TGFBR2-dependent transcriptional upregulation causing increased intracellular and extracellular protein levels. As a new TGFBR2 target gene it may provide a link between the TGF-ß branch and the BMP/GDF branch of SMAD-mediated signaling.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 2
    In: Protein Science, October 2015, Vol.24(10), pp.1686-1694
    Description: Protein‐linked glycans play key roles in cell differentiation, cell–cell interactions, cell growth, adhesion and immune response. Aberrant glycosylation is a characteristic feature of tumor cells and is involved in tumor growth, escape from apoptosis, metastasis formation, and resistance to therapy. It can serve as cancer biomarker and treatment target. To enable comprehensive screening for the impact of tumor driving mutations in colorectal cancer cells we present a method for specific analysis of tumor driver‐induced glycome changes. The strategy is based on a combination of three technologies, that is recombinase‐mediated cassette exchange (RMCE), Click‐It chemistry and mass spectrometry. The new method is exemplified by the analysis of the impact of inactivating mutations of the TGF‐ß‐receptor type II (TGFBR2) on sialic acid incorporation into protein‐linked glycans of the colon cancer cell line HCT116. Overall, 70 proteins were found to show sialic acid incorporation exclusively upon TGFBR2 expression whereas 7 proteins lost sialylation upon TGFBR2 reconstitution. Validation of detected candidate glycoproteins is demonstrated with the cell surface glycoprotein nectin‐3 known to be involved in metastasis, invasion and prognosis of various cancers. Altogether, our new approach can help to systematically puzzle out the influence of tumor‐specific mutations in a major signaling pathway, as exemplified by the TGFBR2 tumor suppressor, on the tumor glycome. It facilitates the identification of glycan‐based tumor markers that could be used for diagnostic and therapeutic applications. In principle the outlined strategy can be adapted to any cancer cell line, tumor driver mutation and several glycan‐building blocks.
    Keywords: Colorectal Cancer ; Microsatellite Instability ; Sialylation ; Tgfbr2 ; Nectin‐3
    ISSN: 0961-8368
    E-ISSN: 1469-896X
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  • 3
    Language: English
    In: Molecular & cellular proteomics : MCP, December 2014, Vol.13(12), pp.3446-56
    Description: Activin receptor type II (ACVR2) is a member of the transforming growth factor type II receptor family and controls cell growth and differentiation, thereby acting as a tumor suppressor. ACVR2 inactivation is known to drive colorectal tumorigenesis. We used an ACVR2-deficient microsatellite unstable colon cancer cell line (HCT116) to set up a novel experimental design for comprehensive analysis of proteomic changes associated with such functional loss of a tumor suppressor. To this end we combined two existing technologies. First, the ACVR2 gene was reconstituted in an ACVR2-deficient colorectal cancer (CRC) cell line by means of recombinase-mediated cassette exchange, resulting in the generation of an inducible expression system that allowed the regulation of ACVR2 gene expression in a doxycycline-dependent manner. Functional expression in the induced cells was explicitly proven. Second, we used the methionine analog azidohomoalanine for metabolic labeling of newly synthesized proteins in our cell line model. Labeled proteins were tagged with biotin via a Click-iT chemistry approach enabling specific extraction of labeled proteins by streptavidin-coated beads. Tryptic on-bead digestion of captured proteins and subsequent ultra-high-performance LC coupled to LTQ Orbitrap XL mass spectrometry identified 513 proteins, with 25 of them differentially expressed between ACVR2-deficient and -proficient cells. Among these, several candidates that had already been linked to colorectal cancer or were known to play a key role in cell growth or apoptosis control were identified, proving the utility of the presented experimental approach. In principle, this strategy can be adapted to analyze any gene of interest and its effect on the cellular de novo proteome.
    Keywords: Gene Expression Regulation, Neoplastic ; Activin Receptors, Type II -- Genetics ; Proteome -- Genetics ; Staining and Labeling -- Methods
    ISSN: 15359476
    E-ISSN: 1535-9484
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  • 4
    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 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. Graphical Abstract 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.
    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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  • 5
    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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  • 6
    Language: English
    In: Experimental Cell Research
    Description: Galectins are β-galactoside binding proteins which possess a variety of functions including modulation of apoptosis, growth and differentiation. Hence, alterations in the expression profile have been associated with loss of cellular homeostasis contributing to tumor growth and progression. Though galectin-12 is significantly downregulated in several tumor entities, including colon cancer, its impact on cellular homeostasis as well as galectin-12 specific binding partners have not been identified so far. We therefore established an experimental strategy which is based on reversible cross-link immunoprecipitation to capture the galectin-12 protein interactome in colon cancer cells. By applying this approach, we identified 10 novel candidates of galectin-12 interacting proteins including the neutral amino acid exchanger SLC1A5. Remarkably, we uncovered that binding of galectin-12 to SLC1A5 significantly reduced glutamine uptake in our model cell line. Consequently, utilization of glutamine carbon for biomass synthesis was profoundly affected, suggesting galectin-12 as a novel inhibitor of glutamine anaplerosis in colon cancer cells. More detailed analysis revealed that colon cancer cells can counteract galectin-12 mediated glutamine deprivation by induction of compensatory mechanisms which facilitate adaption to low-glutamine conditions and thus survival.
    Keywords: Colorectal Cancer ; Galectins ; Proteomics ; Cross-Linking ; Metabolism ; Glutaminolysis ; Biology
    ISSN: 0014-4827
    E-ISSN: 1090-2422
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  • 7
    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 extracellular vesicles (EVs). EVs function as couriers of proteins, nucleic acids, and lipids in intercellular communication. At a qualitative level, we have previously shown that TGFBR2 deficiency causes overall alterations in the EV protein content. To deepen the basic understanding of altered protein dynamics, this work aimed to determine TGFBR2-dependent EV protein signatures in a quantitative manner. Using a stable isotope labeling with amino acids in cell culture (SILAC) approach for mass spectrometry-based quantification, 48 TGFBR2-regulated proteins were identified in MSI CRC-derived EVs. Overall, TGFBR2 deficiency caused upregulation of several EV proteins related to the extracellular matrix and nucleosome as well as downregulation of proteasome-associated proteins. The present study emphasizes the general overlap of proteins between EVs and their parental CRC cells but also highlights the impact of TGFBR2 deficiency on EV protein composition. From a clinical perspective, TGFBR2-regulated quantitative differences of protein expression in EVs might nominate novel biomarkers for liquid biopsy-based MSI typing in the future.
    Keywords: Extracellular Vesicles ; Exosomes ; Proteomics ; Stable Isotope Labeling With Amino Acids in Cell Culture (Silac) ; Tgfbr2 ; Microsatellite Instability ; DNA Mismatch Repair ; Colorectal Cancer ; Biology
    E-ISSN: 1422-0067
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