title: Transforming growth factor beta receptor 2 (TGFBR2) changes sialylation in the microsatellite unstable (MSI) Colorectal cancer cell line HCT116.
abstract: Aberrant glycosylation is a common feature of many malignancies including colorectal cancers (CRCs). About 15% of CRC show the microsatellite instability (MSI) phenotype that is associated with a high frequency of biallelic frameshift mutations in the A10 coding mononucleotide microsatellite of the transforming growth factor beta receptor 2 (TGFBR2) gene. If and how impaired TGFBR2 signaling in MSI CRC cells affects cell surface glycan pattern is largely unexplored. Here, we used the TGFBR2-deficient MSI colon carcinoma cell line HCT116 as a model system. Stable clones conferring doxycycline (dox)-inducible expression of a single copy wildtype TGFBR2 transgene were generated by recombinase-mediated cassette exchange (RMCE). In two independent clones, dox-inducible expression of wildtype TGFBR2 protein and reconstitution of its signaling function was shown. Metabolic labeling experiments using the tritiated sialic acid precursor N-acetyl-D-mannosamine (ManNAc) revealed a significant decline (∼30%) of its incorporation into newly synthesized sialoglycoproteins in a TGFBR2-dependent manner. In particular, we detected a significant decrease of sialylated ß1-integrin upon reconstituted TGFBR2 signaling which did not influence ß1-integrin protein turnover. Notably, TGFBR2 reconstitution did not affect the transcript levels of any of the known human sialyltransferases when examined by real-time RT- PCR analysis. These results suggest that reconstituted TGFBR2 signaling in an isogenic MSI cell line model system can modulate sialylation of cell surface proteins like ß1-integrin. Moreover, our model system will be suitable to uncover the underlying molecular mechanisms of altered MSI tumor glycobiology.
authors.affiliation: Department of Applied Tumor Biology, Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lee
authors.forename: Seda
authors.initials: S
authors.lastname: Ballikaya
authors.forename: Kai
authors.initials: K
authors.lastname: Schönig
authors.forename: Claudia R
authors.initials: CR
authors.lastname: Ball
authors.forename: Hanno
authors.initials: H
authors.lastname: Glimm
authors.forename: Juergen
authors.initials: J
authors.lastname: Kopitz
authors.forename: Johannes
authors.initials: J
authors.lastname: Gebert
mesh_terms: D001483:Base Sequence; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism*; D004318:Doxycycline / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D023061:Gene Order; D018390:Gene Targeting; D042822:Genomic Instability; D045325:HCT116 Cells; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism; D018895:Microsatellite Repeats; D011134:Polysaccharides / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*
chemical_list: D019012:Integrin beta1; D011134:Polysaccharides; D018125:Receptors, Transforming Growth Factor beta; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D004318:Doxycycline