Transcriptional activation of fucosyltransferase (FUT) genes using the CRISPR-dCas9-VPR technology reveals potent N-glycome alterations in colorectal cancer cells

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 well-known 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 core-fucosylation, 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.

MiR-29b/Sp1/FUT4 axis modulates the malignancy of leukemia stem cells by regulating fucosylation via Wnt/β-catenin pathway in acute myeloid leukemia

“…Fucosylation is an important functional regulation of glycoproteins and is critical for the abnormal glycosylation during tumor progression. FUT4 is abnormally upregulated in different types of cancers, such as colorectal cancer [19], breast cancer [20] and lung adenocarcinoma [21], and the up-regulation of FUT4 is associated with tumor metastases, higher recurrence, and poorer survival in tumor patients. According to these findings, we speculate that FUT4 is a key factor in promoting malignant progression of tumors.…”

miR-200c /FUT4 axis prevents the migration, invasion and proliferation of colon cancer cells by downregulating Wnt/β-catenin pathway

“…FUT family is a class of glycosyltransferase molecules that are involved in the synthesis of glycoproteins and glycolipid sugar chains on the cell surface, which play important role in a variety of physiological processes. 19 The FUT family can be divided into four subfamilies according to the different transferase activities. FUT4 is a key enzyme for the synthesis of sialylated Lewis oligosaccharide X (SLeX), and the previous study showed that abnormal expression of FUT4 has important links with tumorigenesis, invasion and metastasis.…”

OGT Controls the Expression and the Glycosylation of E‐cadherin, and Affects Glycosphingolipid Structures in Human Colon Cell Lines

“…Samples were vortexed and then incubated overnight at 37 °C. The released glycans were then converted to aldoses with 0.1% formic acid, filtered through a protein‐binding membrane, and dried . Released N ‐glycans were fluorescently labelled by reductive amination with procainamide using LudgerTag Procainamide Glycan Labelling Kit (LT‐KPROC‐24) as described previously…”

“…Procainamide‐labelled N ‐glycan samples and system suitability standards were analyzed by HILIC‐(U)HPLC‐ESI‐MS with fluorescence detection, using the previously described protocol . Relative glycan quantification was achieved using HappyTools software, allowing automated peak, background, and noise detection .…”