Transforming growth factor beta receptor 2 (TGFBR2) changes sialylation in the microsatellite unstable (MSI) Colorectal cancer cell line HCT116

PLoS One. 2013;8(2):e57074. doi: 10.1371/journal.pone.0057074. Epub 2013 Feb 27.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Colorectal Neoplasms / genetics*
  • Colorectal Neoplasms / metabolism*
  • Doxycycline / pharmacology
  • Gene Expression Regulation / drug effects
  • Gene Order
  • Gene Targeting
  • Genomic Instability*
  • HCT116 Cells
  • Humans
  • Integrin beta1 / metabolism
  • Microsatellite Repeats*
  • Polysaccharides / metabolism
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Receptor, Transforming Growth Factor-beta Type II
  • Receptors, Transforming Growth Factor beta / genetics
  • Receptors, Transforming Growth Factor beta / metabolism*

Substances

  • Integrin beta1
  • Polysaccharides
  • Receptors, Transforming Growth Factor beta
  • Protein-Serine-Threonine Kinases
  • Receptor, Transforming Growth Factor-beta Type II
  • Doxycycline

Grant support

Financial support was provided by DFG (GE592/6-1) to J.K. and J.G. and DFG (KFO 227) to C.R.B. and H.G. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.