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.
Highlights
About 15% of hereditary and sporadic colorectal tumors display the high frequency microsatellite instability phenotype
In previous work we have demonstrated that transient transfection of transforming growth factor beta receptor 2 (TGFBR2) into HCT116 cells, deficient for this receptor, led to alterations of cell surface glycosylation in these microsatellite instability (MSI) tumor cells [30]
In order to overcome the transient nature of this expression system and to allow for a more detailed analysis of these TGFBR2mediated glycan alterations and its associated downstream signaling effects, we have generated the HCT116-TGFBR2 MSI tumor cell line model system
Summary
About 15% of hereditary and sporadic colorectal tumors display the high frequency microsatellite instability ( termed MSI) phenotype. Upon binding of the ligand TGF-ß1 the formation of a hetero-tetrameric receptor complex comprised of type 1 (TGFBR1) and type 2 (TGFBR2) receptors is initiated and downstream proteins such as SMAD2 and SMAD3 become phosphorylated [10,12]. These phosphorylated SMAD proteins associate with SMAD4 and upon translocation to the nucleus direct transcription of different target genes like SMAD7 [13] or SERPINE [14]
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