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 Lewis^x 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 Lewis^x 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 Lewis^x antigen on the cell surface. Interestingly, Lewis^x 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, Lewis^x 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.

中文翻译：

---

使用CRISPR-dCas9-VPR技术的岩藻糖基转移酶（FUT）基因的转录激活揭示了结直肠癌细胞中的强力N糖基改变

癌细胞中的岩藻糖基化异常被认为是恶性细胞转化的标志，它与肿瘤的进展，转移和对化疗的耐药性有关。具体而言，在结直肠癌细胞中，岩藻糖基化的Lewis ^x抗原水平的升高归因于相关岩藻糖基转移酶（例如岩藻糖基转移酶4（FUT4）和岩藻糖基转移酶9（FUT9））的表达失调。然而，到目前为止，缺乏紧密模拟类似于岩藻糖基转移酶基因表达的癌症特异性调控的实验模型，迄今为止，我们对这些酶的底物特异性以及Lewis ^x合成对结直肠癌细胞糖基化的影响的认识有限。因此，我们试图转录激活Fut4和著名的小鼠结肠直肠癌细胞系MC38中的Fut9和Fut9基因缺乏FUT4和FUT9酶的表达。为了这个目的，我们利用了从生理上相关的，基于指导RNA的从头基因表达的模型，即CRISPR-dCas9-VPR系统。使用CRISPR-dCas9-VPR诱导MC38细胞中的Fut4和Fut9基因在细胞表面特异性表达功能性Lewis ^x抗原。有趣的是，尽管在生物合成特性和诱导的酶的表达稳定性方面存在明显差异，路易斯^x仍主要由MC 38-FUT4和MC38-FUT9细胞中的N-连接聚糖所携带。此外，刘易斯（Lewis）^x发现其表达影响MC38-糖变异体的核心岩藻糖基化，唾液酸化，触角和N-聚糖亚型。总之，利用CRISPR-dCas9-VPR系统增强糖基转移酶的表达是一种有前途的转录基因激活方法，在糖生物学和肿瘤学研究中具有广泛的应用可能性。