Altered N-Glycan Expression Profile in Epithelial-to-Mesenchymal Transition of NMuMG Cells Revealed by an Integrated Strategy Using Mass Spectrometry and Glycogene and Lectin Microarray Analysis

Abstract

Epithelial-to-mesenchymal transition (EMT) is an essential biological process that occurs in embryonic development, metastatic diseases, and cancer progression. Altered expression of glycans is known to be associated with cancer progression. No studies to date have presented global analysis of the precise variation of N-glycans in EMT. We describe here the profile of N-glycans and glycogene expression in the EMT process induced by transforming growth factor-β1 (TGFβ1) in a normal mouse mammary gland epithelial (NMuMG) cell model. An integrated strategy with a combination of mass spectrometry, glycogene microarray analysis, and lectin microarray analysis was applied, and results were confirmed by lectin histochemistry and quantitative real-time PCR. In TGFβ-induced EMT, levels of high-mannose-type N-glycans were enhanced, antennary N-glycans, and fucosylation were suppressed, and bisecting GlcNAc N-glycans were greatly suppressed. The expression of seven N-glycan-related genes was significantly changed. The products of glycogenes ALG9, MGAT3, and MGAT4B appeared to contribute to the observed alteration of N-glycans. The findings indicate that dysregulation of N-glycan synthesis plays a role in the EMT process. Systematic glycomic analysis based on the combination of techniques described here is expected to facilitate the discovery of the aberrant N-glycosylation in tumor progression and provide essential information in systems glycobiology.

β-Galactoside α2,6-Sialyltranferase 1 Promotes Transforming Growth Factor-β-mediated Epithelial-Mesenchymal Transition

“…The phosphorylated Smad proteins then form a trimer with Smad4 and translocate into the nucleus, where they associate and cooperate with other transcription factors to precisely regulate the target gene transcription (10). These reorchestrated target genes, as recently evidenced in several types of cancer cell lines undergoing EMT by mass spectrometry and microarray analysis, at least include the glycogenes involved in N-glycosylation, O-glycosylation, and sialylation (11)(12)(13), indicating the importance of cellular glycosylation pattern in both the transition to and the maintenance of the mesenchymal state.…”

Glycans and Cancer

“…Moreover, glycosylation plays a pivotal role in cancer progression and metastasis, cell-cell contact, and epithelial-mesenchymal transition (EMT) in cancer cells (Chen et al, 2013;Kalluri & Weinberg, 2009;Li et al, 2014;Pinho et al, 2012;Tan et al, 2014;Terao et al, 2011;Xu et al, 2012). In recent years, EMT has become the important issue for understanding the development and metastasis of cancer (Kalluri & Weinberg, 2009), and in fact, changes in N-glycan structures are considered to be important for understanding the significance of EMT and the resultant change of adhesive properties of cancer cells (Chen et al, 2013;Li et al, 2014;Pinho et al, 2012;Tan et al, 2014;Terao et al, 2011;Xu et al, 2012).…”

Quantitative analysis of glycans, related genes, and proteins in two human bone marrow stromal cell lines using an integrated strategy

“…On the other hand, fluorescence intensity of LEL, which could also recognized Galβ1,4GlcNAc, was lower in HS27a. This mainly because that the glycan-binding specificities of many lectins remain to be fully explored [26], and differences in structure need to be confirmed by mass spectroscopic and other techniques. LacNAc synthesis is part of the pathway leading to the terminal capping group sialyl Lewis X (Le x ) [53, 54].…”

“…Open-access data at http://www.ncbi.nlm.nih.gov/sites/GDSbrowser (GEO accession: GSE463) were downloaded. Glycogenes listed on the GlycoV4 oligonucleotide microarray (covering 1260 human glycogenes) were extracted, and analyzed as described [26, 27]. Raw values were normalized using the robust multichip average (RMA) expression summary.…”