Abstract
Aberrant fucosylation in cancer cells is considered as a signature of malignant cell transformation
and it is associated with tumor progression, metastasis and resistance to chemotherapy. Specifically,
in colorectal cancer cells, increased levels of the fucosylated Lewisx antigen are attributed to the
deregulated expression of pertinent fucosyltransferases, like fucosyltransferase 4 (FUT4) and fucosyltransferase
9 (FUT9). However, the lack of experimental models closely mimicking cancer-specific
regulation of fucosyltransferase gene expression has, so far, limited our knowledge regarding the
substrate specificity of these enzymes and the impact of Lewisx synthesis on the glycome of colorectal
cancer cells. Therefore, we sought to transcriptionally activate the Fut4 and Fut9 genes in the wellknown
murine colorectal cancer cell line, MC38, which lacks expression of the FUT4 and FUT9
enzymes. For this purpose, we utilized a physiologically relevant, guide RNA-based model of de novo
gene expression, namely the CRISPR-dCas9-VPR system. Induction of the Fut4 and Fut9 genes in
MC38 cells using CRISPR-dCas9-VPR resulted in specific neo-expression of functional Lewisx antigen
on the cell surface. Interestingly, Lewisx was mainly carried by N-linked glycans in both MC38-FUT4
and MC38-FUT9 cells, despite pronounced differences in the biosynthetic properties and the expression
stability of the induced enzymes. Moreover, Lewisx expression was found to influence corefucosylation,
sialylation, antennarity and the subtypes of N-glycans in the MC38-glycovariants. In
conclusion, exploiting the CRISPR-dCas9-VPR system to augment glycosyltransferase expression is
a promising method of transcriptional gene activation with broad application possibilities in glycobiology
and oncology research.
and it is associated with tumor progression, metastasis and resistance to chemotherapy. Specifically,
in colorectal cancer cells, increased levels of the fucosylated Lewisx antigen are attributed to the
deregulated expression of pertinent fucosyltransferases, like fucosyltransferase 4 (FUT4) and fucosyltransferase
9 (FUT9). However, the lack of experimental models closely mimicking cancer-specific
regulation of fucosyltransferase gene expression has, so far, limited our knowledge regarding the
substrate specificity of these enzymes and the impact of Lewisx synthesis on the glycome of colorectal
cancer cells. Therefore, we sought to transcriptionally activate the Fut4 and Fut9 genes in the wellknown
murine colorectal cancer cell line, MC38, which lacks expression of the FUT4 and FUT9
enzymes. For this purpose, we utilized a physiologically relevant, guide RNA-based model of de novo
gene expression, namely the CRISPR-dCas9-VPR system. Induction of the Fut4 and Fut9 genes in
MC38 cells using CRISPR-dCas9-VPR resulted in specific neo-expression of functional Lewisx antigen
on the cell surface. Interestingly, Lewisx was mainly carried by N-linked glycans in both MC38-FUT4
and MC38-FUT9 cells, despite pronounced differences in the biosynthetic properties and the expression
stability of the induced enzymes. Moreover, Lewisx expression was found to influence corefucosylation,
sialylation, antennarity and the subtypes of N-glycans in the MC38-glycovariants. In
conclusion, exploiting the CRISPR-dCas9-VPR system to augment glycosyltransferase expression is
a promising method of transcriptional gene activation with broad application possibilities in glycobiology
and oncology research.
| Original language | English |
|---|---|
| Pages (from-to) | 137-150 |
| Journal | Glycobiology |
| Volume | 29 |
| Issue number | 2 |
| Early online date | 16 Oct 2018 |
| DOIs | |
| Publication status | Published - 1 Feb 2019 |