Reconstitution of TGFBR2 in HCT116 colorectal cancer cells causes increased LFNG expression and enhanced N-acetyl-d-glucosamine incorporation into Notch1

Cell Signal. 2016 Aug;28(8):1105-13. doi: 10.1016/j.cellsig.2016.04.012. Epub 2016 May 5.

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.

Keywords: Colorectal cancer; Glycosylation; LFNG; Microsatellite instability; Notch1; TGFBR2.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine / metabolism*
  • Blotting, Western
  • Cell Movement / genetics
  • Cell Proliferation
  • Clone Cells
  • Colorectal Neoplasms / genetics*
  • Colorectal Neoplasms / pathology
  • Gene Expression Regulation, Neoplastic*
  • Glycomics
  • Glycosylation
  • Glycosyltransferases / genetics*
  • Glycosyltransferases / metabolism
  • HCT116 Cells
  • Hep G2 Cells
  • Humans
  • Oligonucleotide Array Sequence Analysis
  • Protein-Serine-Threonine Kinases / metabolism*
  • Real-Time Polymerase Chain Reaction
  • Receptor, Transforming Growth Factor-beta Type II
  • Receptors, Notch / metabolism*
  • Receptors, Transforming Growth Factor beta / metabolism*
  • Reproducibility of Results

Substances

  • Receptors, Notch
  • Receptors, Transforming Growth Factor beta
  • Glycosyltransferases
  • LFNG protein, human
  • Protein-Serine-Threonine Kinases
  • Receptor, Transforming Growth Factor-beta Type II
  • Acetylglucosamine