title: Reconstitution of TGFBR2 in HCT116 colorectal cancer cells causes increased LFNG expression and enhanced N-acetyl-d-glucosamine incorporation into Notch1.
abstract: Transforming growth factor-β (TGF-β) signaling plays a key role in regulating normal cell growth and differentiation, and mutations affecting members of this pathway contribute to cancer development and metastasis. In DNA mismatch repair (MMR)-deficient colorectal cancers that exhibit the microsatellite instability (MSI) phenotype, biallelic frameshift mutations in the transforming growth factor β receptor type 2 (TGFBR2) gene occur at high frequency that lead to altered signal transduction and downstream target gene expression. Although recent evidence suggests that altered TGF-β signaling can modulate protein glycosylation patterns in MSI-high colorectal tumor cells, affected genes have not been identified. Here, we investigated in a more systematic approach, expression changes of TGFBR2-regulated genes, involved in glycosylation using a TGFBR2-reconstituted colorectal cancer cell line (HCT116-TGFBR2) and Glyco-Gene Chip analysis. Based on this oligonucleotide array of about 1000 human glycosylation-related genes, several candidates including HES1, PDGFB, JUNB and LFNG were found to be upregulated in a TGFBR2-dependent manner and subsequently validated by real-time RT-PCR analyses. Focusing on the glycosyltransferase LFNG and its target signaling protein Notch1, dual labeling with [3H]-N-acetyl-d-glucosamine ([3H]-GlcNAc) and [35S]-l-methionine revealed a significant increase in N-acetyl-d-glucosamine incorporation into immunoprecipitated Notch1 receptor upon TGFBR2 expression whereas the protein level remained unaffected. These data suggest that TGFBR2 signaling can affect Notch1 glycosylation via regulation of glycosyltransferase LFNG expression and provide a first mechanistic example for altered glycosylation in MSI colorectal tumor cells.
authors.affiliation: Department of Applied Tumor Biology, Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany; Cancer Early Detection, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Applied Tumor Biology, Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany; Cancer Early Detection, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Eva-Maria
authors.initials: EM
authors.lastname: Katzenmaier
authors.affiliation: Department of Applied Tumor Biology, Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany; Cancer Early Detection, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Kopitz
authors.affiliation: Department of Applied Tumor Biology, Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany; Cancer Early Detection, German Cancer Research Center (DKFZ), Heidelberg, Germany. Electronic address: Johannes.Gebert@med.uni-heidelberg.de.
authors.forename: Johannes
authors.initials: J
authors.lastname: Gebert
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D015153:Blotting, Western; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D002999:Clone Cells; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D054794:Glycomics; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics* / Q000378:metabolism; D045325:HCT116 Cells; D056945:Hep G2 Cells; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D000077294:Receptor, Transforming Growth Factor-beta Type II; D051880:Receptors, Notch / Q000378:metabolism*; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism*; D015203:Reproducibility of Results
chemical_list: D051880:Receptors, Notch; D018125:Receptors, Transforming Growth Factor beta; D016695:Glycosyltransferases; C498393:LFNG protein, human; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D000117:Acetylglucosamine
keywords: Colorectal cancer; Glycosylation; LFNG; Microsatellite instability; Notch1; TGFBR2