Glycosylation is a common posttranslational modification on membrane-associated and secreted proteins that is of pivotal importance for regulating cell functions. Aberrant glycosylation can lead to uncontrolled cell proliferation, cell-matrix interactions, migration and differentiation, and has been shown to be involved in cancer and other diseases. The epithelial-to-mesenchymal transition is a key step in the metastatic process by which cancer cells gain the ability to invade tissues and extravasate into the bloodstream. This cellular transformation process, which is associated by morphological change, loss of epithelial traits and gain of mesenchymal markers, is triggered by the secreted cytokine transforming growth factor-β (TGF-β). TGF-β bioactivity is carefully regulated, and its effects on cells are mediated by its receptors on the cell surface. In this review, we first provide a brief overview of major types of glycans, namely, N-glycans, O-glycans, glycosphingolipids and glycosaminoglycans that are involved in cancer progression. Thereafter, we summarize studies on how the glycosylation of TGF-β signaling components regulates TGF-β secretion, bioavailability and TGF-β receptor function. Then, we review glycosylation changes associated with TGF-β-induced epithelial-to-mesenchymal transition in cancer. Identifying and understanding the mechanisms by which glycosylation affects TGF-β signaling and downstream biological responses will facilitate the identification of glycans as biomarkers and enable novel therapeutic approaches.

Changes in glycosylation signatures of cells have been associated with pathological processes in cancer as well as infectious and autoimmune diseases. The current protocols for comprehensive analysis of N-glycomics and O-glycomics derived from cells and tissues often require a large amount of biological material. They also only allow the processing of very limited numbers of samples at a time. Here we established a workflow for sequential release of N-glycans and O-glycans based on PVDF membrane immobilization in 96-well format from 5 × 10⁵ cells. Released glycans are reduced, desalted, purified, and reconstituted, all in 96-well format plates, without additional staining or derivatization. Glycans are then analyzed with porous graphitized carbon nano-liquid chromatography coupled to tandem mass spectrometry using negative-mode electrospray ionization, enabling the chromatographic resolution and structural elucidation of glycan species including many compositional isomers. The approach was demonstrated using glycoprotein standards and further applied to analyze the glycosylation of the murine mammary gland NMuMG cell line. The developed protocol allows the analysis of N- and O-glycans from relatively large numbers of samples in a less time consuming way with high repeatability. Inter- and intraday repeatability of the fetuin N-glycan analysis showed two median intraday coefficients of variations (CVs) of 7.6% and 8.0%, and a median interday CV of 9.8%. Median CVs of 7.9% and 8.7% for the main peaks of N- and O-glycans released from the NMuMG cell line indicate a very good repeatability. The method is applicable to purified glycoproteins as well as to biofluids and cell- or tissue-based samples.

Epithelial‐mesenchymal transition (EMT) is pivotal in the initiation and development of cancer cell metastasis. We observed that the abundance of glycosphingolipids (GSLs), especially ganglioside subtypes, decreased significantly during TGF‐β‐induced EMT in NMuMG mouse mammary epithelial cells and A549 human lung adenocarcinoma cells. Transcriptional profiling showed that TGF‐β/SMAD response genes and EMT signatures were strongly enriched in NMuMG cells, along with depletion of UDP‐glucose ceramide glucosyltransferase (UGCG), the enzyme that catalyzes the initial step in GSL biosynthesis. Consistent with this finding, genetic or pharmacological inhibition of UGCG promoted TGF‐β signaling and TGF‐β‐induced EMT. UGCG inhibition promoted A549 cell migration, extravasation in the zebrafish xenograft model, and metastasis in mice. Mechanistically, GSLs inhibited TGF‐β signaling by promoting lipid raft localization of the TGF‐β type I receptor (TβRI) and by increasing TβRI ubiquitination and degradation. Importantly, we identified ST3GAL5‐synthesized a‐series gangliosides as the main GSL subtype involved in inhibition of TGF‐β signaling and TGF‐β‐induced EMT in A549 cells. Notably, ST3GAL5 is weakly expressed in lung cancer tissues compared to adjacent nonmalignant tissues, and its expression correlates with good prognosis.

The human Gb3/CD77 synthase, encoded by the A4GALT gene, is an unusually promiscuous glycosyltransferase. It synthesizes the Galα1→4Gal linkage on two different glycosphingolipids (GSLs), producing globotriaosylceramide (Gb3, CD77, Pk) and the P1 antigen. Gb3 is the major receptor for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli. A single amino acid substitution (p.Q211E) ramps up the enzyme's promiscuity, rendering it able to attach Gal both to another Gal residue and to GalNAc, giving rise to NOR1 and NOR2 GSLs. Human Gb3/CD77 synthase was long believed to transfer Gal only to GSL acceptors, therefore its GSL products were, by default, considered the only human Stx receptors. Here, using soluble, recombinant human Gb3/CD77 synthase and p.Q211E mutein, we demonstrate that both enzymes can synthesize the P1 glycotope (terminal Galα1→4Galβ1→4GlcNAc-R) on a complex type N-glycan and a synthetic N-glycoprotein (saposin D). Moreover, by transfection of CHO-Lec2 cells with vectors encoding human Gb3/CD77 synthase and its p.Q211E mutein, we demonstrate that both enzymes produce P1 glycotopes on N-glycoproteins, with the mutein exhibiting elevated activity. These P1-terminated N-glycoproteins are recognized by Stx1 but not Stx2 B subunits. Finally, cytotoxicity assays show that Stx1 can use P1 N-glycoproteins produced in CHO-Lec2 cells as functional receptors. We conclude that Stx1 can recognize and use P1 N-glycoproteins in addition to its canonical GSL receptors to enter and kill the cells, while Stx2 can use GSLs only. Collectively, these results may have important implications for our understanding of the Shiga toxin pathology.

Acute myeloid leukemia (AML) is characterized by a dysregulated expansion of poorly differentiated myeloid cells. Although patients are usually treated effectively by chemotherapy, a high rate of relapsed or refractory disease poses a major hurdle in its treatment. Recently, several studies have proposed implications of protein glycosylation in the pathobiology of AML including chemoresistance. Accordingly, associations have been found between specific glycan epitopes and the outcome of the disease. To advance this poorly studied field, we performed an exploratory glycomics study characterizing 21 widely used AML cell lines. Exploiting the benefits of porous graphitized carbon chromatography coupled to tandem mass spectrometry (PGC nano-LC-MS²), we qualitatively and quantitatively profiled N- and O-linked glycans. AML cell lines exhibited distinct glycan fingerprints differing in relevant glycan traits correlating with their cellular phenotype as classified by the FAB system. By implementing transcriptomics data, specific glycosyltransferases and hematopoietic transcription factors were identified, which are candidate drivers of the glycan phenotype of these cells. In conclusion, we report the varying expression of glycan structures across a high number of AML cell lines, including those associated with poor prognosis, identified underlying glycosyltransferases and transcription factors, and provide insights into the regulation of the AML glycan repertoire.