Biochemical and functional characterization of glycosylation-associated mutational landscapes in colon cancer

Abstract

The molecular basis of aberrant protein glycosylation, a pathological alteration widespread in colorectal cancers (CRC), and the mechanisms by which it contributes to tumor progression remain largely unknown. We performed targeted re-sequencing of 430 glycosylation-associated genes in a series of patient-derived CRC cell lines (N = 31) and matched primary tumor tissues, identifying 12 new significantly mutated glycosylation-associated genes in colon cancer. In particular, we observed an enrichment of mutations in genes (B3GNT2, B4GALT2, ST6GALNAC2) involved in the biosynthesis of N- and Cores 1–3 O-linked glycans in the colon, accounting for ~16% of the CRCs tested. Analysis of independent large-scale tumor tissue datasets confirmed recurrent mutations within these genes in colon and other gastrointestinal cancers. Systematic biochemical and phenotypic characterization of the candidate wild-type and mutant glycosyltransferases demonstrated these mutations as either markedly altering protein localization, post-translational modification, encoded enzymatic activities and/or the migratory potential of colon carcinoma cells. These findings suggest that functionally deleterious mutations in glycosyltransferase genes in part underlie aberrant glycosylation, and contribute to the pathogenesis of molecular subsets of colon and other gastrointestinal malignancies.

Altered glycosyltransferases in colorectal cancer

“…In particular, our previous studies in familial and sporadic colorectal cancers (CRCs) identified functionally-inactivating germline and somatic mutations in the gene encoding for GALNT12 (Polypeptide N-acetylgalactosaminyltransferase 12) [7], suggesting that aberrant glycosylation in cancers could stem from genetic defects in the glycosyltransferase genes. These findings prompted us to undertake an in-depth characterization of CRC-associated mutational alterations in genes encoding glycosylation enzymes using targeted next-generation sequencing [8]. Of the 430 glycosylation-associated genes assessed, 36 genes exhibited mutational alterations (missense, nonsense, splice site and frameshift mutations) in colorectal cancers, with 12 genes displaying significantly higher mutation rates than the expected background rate in CRCs [8].…”

“…These findings prompted us to undertake an in-depth characterization of CRC-associated mutational alterations in genes encoding glycosylation enzymes using targeted next-generation sequencing [8]. Of the 430 glycosylation-associated genes assessed, 36 genes exhibited mutational alterations (missense, nonsense, splice site and frameshift mutations) in colorectal cancers, with 12 genes displaying significantly higher mutation rates than the expected background rate in CRCs [8]. Interestingly, further enrichment analysis of the 12 genes showed that three genes, B3GNT2 (β-1,3-N Acetylglucosaminyltransferase 2), B4GALT2 (β-1,4-Galactosyltransferase 2) and ST6GALNAC2 (α-N-Acetylgalactosaminidyl α-2,6-Sialyltransferase 2), belonged to polylactosamine and N- and O-glycosylation pathway [8].…”

“…Of the 430 glycosylation-associated genes assessed, 36 genes exhibited mutational alterations (missense, nonsense, splice site and frameshift mutations) in colorectal cancers, with 12 genes displaying significantly higher mutation rates than the expected background rate in CRCs [8]. Interestingly, further enrichment analysis of the 12 genes showed that three genes, B3GNT2 (β-1,3-N Acetylglucosaminyltransferase 2), B4GALT2 (β-1,4-Galactosyltransferase 2) and ST6GALNAC2 (α-N-Acetylgalactosaminidyl α-2,6-Sialyltransferase 2), belonged to polylactosamine and N- and O-glycosylation pathway [8]. While B3GNT2 and B4GALT2 are involved in polyLacNAc biosynthesis on N-linked tetraantennary structures and on Core 1, 2 and 3 O-glycan core structures [9, 10, 11], ST6GALNAC2 terminates glycan chain elongation by the addition of sialic acid to the peptide GalNAc of O-glycan Core 1 or Core 3 structures [10, 12].…”

“…While B3GNT2 and B4GALT2 are involved in polyLacNAc biosynthesis on N-linked tetraantennary structures and on Core 1, 2 and 3 O-glycan core structures [9, 10, 11], ST6GALNAC2 terminates glycan chain elongation by the addition of sialic acid to the peptide GalNAc of O-glycan Core 1 or Core 3 structures [10, 12]. Similar to GALNT12, mutations in these three genes predominantly mapped to the catalytic domain of respective enzymes [8]. Biochemical analyses of wild-type and mutant B3GNT2, ST6GALNAC2, and B4GALT2 demonstrated significant alterations in respective glycosyltransferase function [8].…”

“…Similar to GALNT12, mutations in these three genes predominantly mapped to the catalytic domain of respective enzymes [8]. Biochemical analyses of wild-type and mutant B3GNT2, ST6GALNAC2, and B4GALT2 demonstrated significant alterations in respective glycosyltransferase function [8]. For instance, mutations in B3GNT2 led to either loss of enzymatic function or mislocalization of the protein.…”