Specific glycosphingolipids mediate epithelial-to-mesenchymal transition of human and mouse epithelial cell lines

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The process termed "epithelial-mesenchymal transition" (EMT) was originally discovered in ontogenic development, and has been shown to be one of the key steps in tumor cell progression and metastasis. Recently, we showed that the expression of some glycosphingolipids (GSLs) is down-regulated during EMT in human and mouse cell lines. Here, we demonstrate the involvement of GalNAc-type (or mucintype) O-glycosylation in EMT process, induced with transforming growth factor β (TGF-β) in human prostate epithelial cell lines. We found that: (i) TGF-β treatment caused up-regulation of oncofetal fibronectin (onfFN), which is defined by mAb FDC6, and expressed in cancer or fetal cells/tissues, but not in normal adult cells/tissues. The reactivity of mAb FDC6 requires the addition of an O-glycan at a specific threonine, inside the type III homology connective segment (IIICS) domain of FN. (ii) This change is associated with typical EMT characteristics; i.e., change from epithelial to fibroblastic morphology, enhanced cell motility, decreased expression of a typical epithelial cell marker, E-cadherin, and enhanced expression of mesenchymal markers. (iii) TGF-β treatment up-regulated mRNA level of FN containing the IIICS domain and GalNAc-T activity for the IIICS domain peptide substrate containing the FDC6 onfFN epitope. (iv) Knockdown of GalNAc-T6 and T3 inhibited TGF-β-induced up-regulation of onfFN and EMT process. (v) Involvement of GSLs was not detectable with the EMT process in these cell lines. These findings indicate the important functional role of expression of onfFN, defined by sitespecific O-glycosylation at IIICS domain, in the EMT process.O-glycosylated fibronectin | siRNA G lycoconjugates, such as glycosphingolipids (GSLs) and N-and O-linked glycoproteins, have been shown to play important roles in embryogenesis (1), tumorigenesis, and cancer progression (2). Specific types of glycosyl residues modulate particular signaling pathways and regulate cell phenotypes. For example, GM3 inhibits epithelial growth factor receptor (EGFR) activation (3, 4); GM2 associated with tetraspanine CD82 inhibits the activation of the hepatocyte growth factor receptor cMet (5); and O-Fucose glycans modulate Notch signaling, which controls the fate of many cell types (6-8).Epithelial-mesenchymal transition (EMT) was initially observed during early embryonic development and organ formation (9-11). Accumulating evidence has shown that the EMT process plays a key role in disease development, particularly in cancer progression to metastasis (10-14) and fibrosis (15). During EMT, cells lose their apical-basal polarity, change morphology to fibroblastic, display reduced expression of epithelial cell marker molecules such as E-cadherin (Ecad), and enhance expression of mesenchymal cell marker molecules such as fibronectin (FN), N-cadherin (Ncad), vimentin, and matrix-metalloproteinases (MMPs). When combined, these effects result in increased cell motility (10-15).Our recent studies found a reduction in specific GSLs (Gg4 and/ or GM2) in ...

Section: Discussioncontrasting

confidence: 79%

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confidence: 99%

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confidence: 99%

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Involvement of O-glycosylation defining oncofetal fibronectin in epithelial-mesenchymal transition process

Freire-de-Lima

Gelfenbeyn

Ding

et al. 2011

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113

130

“…This lectin-GSP circuit may be instrumental in maintaining apical transport in MDCK cells. As Guan et al (40) showed in other epithelial cells, different GSPs play a role in maintaining the epithelial state, and this might be attributed to similar lectin-GSP circuits.…”

Section: Discussionmentioning

confidence: 78%

Membrane lipidome of an epithelial cell line

Sampaio

Gerl

Klose

et al. 2011

461

406

Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology to an epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin to glycosphingolipid, together with an increase in plasmalogen, phosphatidylethanolamine, and cholesterol content, whereas the opposite changes took place during an epithelial-to-mesenchymal transition. Moreover, during polarization, the sphingolipids became longer, more saturated, and more hydroxylated as required to generate an apical membrane domain that serves as a protective barrier for the epithelial sheet.epithelial polarity | shotgun lipidomics | high resolution mass spectrometry | Forssman glycosphingolipid | epithelial-mesenchymal transition G ain and loss of epithelial polarity are fundamental processes in tissue differentiation (1, 2). These processes have been extensively studied not only in the context of embryogenesis but in tumor progression and metastasis (1). Although cell polarization necessarily implies major changes in the structure and properties of the cell membranes, the exact changes in the molecular composition of their membrane lipidome remain unknown.Madin-Darby canine kidney (MDCK) cells are a cell culture model for epithelial polarization (3,4). When seeded at low density, MDCK cells undergo a morphological change from a fibroblast-like morphology to a well-polarized epithelium (5) and have recently also been adapted to undergo an epithelial-mesenchymal transition (EMT) (6). Epithelial cells have evolved characteristic apical and basolateral membrane domains, separated by tight junctions, with specific protein and lipid composition (7,8). The unusual robustness of the apical membrane is largely attributable to its special lipid composition (9-11).In this work, we applied shotgun MS for comprehensive and quantitative characterization of mammalian cellular membranes (12). By combining high mass resolution MS, an improved extraction procedure that increases the recovery of polar lipids (13), and the use of internal standard mixtures for absolute quantification, we expanded the lipid repertoire covered by shotgun lipidomics to glycosphingolipids (GSPs). Importantly, the analysis of the GSPs did not require the hydrolysis of glycerophospholipids (GPs) (14), and this simplification allowed us to determine the membrane lipidome with unprecedented comprehensiveness (more than 300 lipid species from 14 different lipid classes) from a single sample of 10 5 cells.We further elucidated how the lipidome is remodeled during the course of MDCK cell epithelial polarization. We found that when these c...

“…The involvement of glycosylation in the EMT process was unclear, particularly in GSLs. Recently, specific GSLs were found to down-regulate during EMT, and enhanced expression of the GSLs blocked the EMT process (22,23).…”

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confidence: 99%

Differential expression profiles of glycosphingolipids in human breast cancer stem cells vs. cancer non-stem cells

Liang

Ding

Levery

et al. 2013

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120

Previous studies demonstrated that certain glycosphingolipids (GSLs) are involved in various cell functions, such as cell growth and motility. Recent studies showed changes in GSL expression during differentiation of human embryonic stem cells; however, little is known about expression profiles of GSLs in cancer stem cells (CSCs). CSCs are a small subpopulation in cancer and are proposed as cancer-initiating cells, have been shown to be resistant to numerous chemotherapies, and may cause cancer recurrence. Here, we analyzed GSLs expressed in human breast CSCs by applying a CSC model induced through epithelial-mesenchymal transition, using mass spectrometry, TLC immunostaining, and cell staining. We found that (i) Fuc-(n)Lc4Cer and Gb3Cer were drastically reduced in CSCs, whereas GD2, GD3, GM2, and GD1a were greatly increased in CSCs; (ii) among various glycosyltransferases tested, mRNA levels for ST3GAL5, B4GALNT1, ST8SIA1, and ST3GAL2 were increased in CSCs, which could explain the increased expression of GD3, GD2, GM2, and GD1a in CSCs; (iii) the majority of GD2+ cells and GD3+ cells were detected in the CD44 hi /CD24 lo cell population; and (iv) knockdown of ST8SIA1 and B4GALNT1 significantly reduced the expression of GD2 and GD3 and caused a phenotype change from CSC to a non-CSC, which was detected by reduced mammosphere formation and cell motility. Our results provide insight into GSL profiles in human breast CSCs, indicate a functional role of GD2 and GD3 in CSCs, and suggest a possible novel approach in targeting human breast CSCs to interfere with cancer recurrence.