A new method for detection of tumor driver‐dependent changes of protein sialylation in a colon cancer cell line reveals nectin‐3 as TGFBR2 target

Abstract

Protein-linked glycans play key roles in cell differentiation, cell-cell interactions, cell growth, adhesion and immune response. Aberrant glycosylation is a characteristic feature of tumor cells and is involved in tumor growth, escape from apoptosis, metastasis formation, and resistance to therapy. It can serve as cancer biomarker and treatment target. To enable comprehensive screening for the impact of tumor driving mutations in colorectal cancer cells we present a method for specific analysis of tumor driver-induced glycome changes. The strategy is based on a combination of three technologies, that is recombinase-mediated cassette exchange (RMCE), Click-It chemistry and mass spectrometry. The new method is exemplified by the analysis of the impact of inactivating mutations of the TGF-ß-receptor type II (TGFBR2) on sialic acid incorporation into protein-linked glycans of the colon cancer cell line HCT116. Overall, 70 proteins were found to show de novo sialic acid incorporation exclusively upon TGFBR2 expression whereas 7 proteins lost sialylation upon TGFBR2 reconstitution. Validation of detected candidate glycoproteins is demonstrated with the cell surface glycoprotein nectin-3 known to be involved in metastasis, invasion and prognosis of various cancers. Altogether, our new approach can help to systematically puzzle out the influence of tumor-specific mutations in a major signaling pathway, as exemplified by the TGFBR2 tumor suppressor, on the tumor glycome. It facilitates the identification of glycan-based tumor markers that could be used for diagnostic and therapeutic applications. In principle the outlined strategy can be adapted to any cancer cell line, tumor driver mutation and several glycan-building blocks.

Glycosylation as a Main Regulator of Growth and Death Factor Receptors Signaling

“…The TGFBR2 genes possess a stretch of 10 adenine residues, which frequently undergo frameshift mutations in MSI CRC, leading to TGFBR2 inactivation, a crucial mutational step for CRC progression. In the CRC cell line HCT116 expressing the biallelic inactivation of TGFBR2, the reconstitution with a wild type TGFBR2 gene leads to altered sialylation of a variety of glycoproteins [ 163 , 164 ], without any change in the expression of sialyltransferases [ 164 ], suggesting a relationship between sialylation and a classical mutational step in CRC. β1,6-branching is involved in the pathogenesis of liver disease through TGFB signaling.…”

Lectin Activity of the TcdA and TcdB Toxins of Clostridium difficile

“…There are also multiple known protein receptors for TcdB. All of the protein targets, NECTIN3 (19), CSPG4 (18, 20), and FZD1/2/7 (20, 21), are glycoproteins that express a range of different glycosylations, including glycosaminoglycans (chondroitin) (18, 20) and complex N-linked glycans (31, 32). A recent study by Chen et al demonstrated the structure of a region of TcdB (TcdB-FZD binding domain [FBD]) responsible for the interaction with FZD proteins outside the tested region of ToxB-B2 (21).…”

TGFBR2‑dependent alterations of microRNA profiles in extracellular vesicles and parental colorectal cancer cells

“…As a consequence, the downstream effects and modifications identified in the EV cargo can be assigned specifically to the TGFBR2 expression status of MSI cells. For example, our previous studies showed that the reconstituted expression of TGFBR2 alters the glycophenotype of MSI tumor cells (15,32,33), and tumor driver-induced glycome changes have been linked to TGFBR2-regulated genes (34). Additionally, the reconstituted expression of TGFBR2 can cause differential de novo protein expression (35).…”