Role of glycosyltransferases in carcinogenesis; growth factor signaling and EMT/MET programs

The glycosylation of cell surface receptors has been shown to regulate each step of signal transduction, including receptor trafficking to the cell surface, ligand binding, dimerization, phosphorylation, and endocytosis. In this review we focus on the role of glycosyltransferases that are involved in the modification of N-glycans, such as the effect of branching and elongation in signaling by various cell surface receptors. In addition, the role of those enzymes in the EMT/MET programs, as related to differentiation and cancer development, progress and therapy resistance is discussed.

Most secreted proteins and membrane proteins in eukaryotic cells are glycosylated, indicating that glycosylation is important for the regulation of signal transduction [1][2][3][4][5][6][7][8]. Glycans control many of the physicochemical properties of proteins, including structure, structural stability, charge, and hydrophilicity. In the case of cell surface receptors, glycosylation has been shown to regulate each step in signal transduction such as receptor trafficking to the cell surface, ligand binding, dimerization, phosphorylation, and endocytosis [9, 10]. To elucidate the mechanisms by which glycans control the function of these receptors, the site and the structure of the responsible glycan(s) need to be determined. To determine the site of the responsible glycan(s), a mutant receptor which lacks specific glycan(s) are used. The N-glycan deleted mutant receptor is established and the effects of the alteration of glycosylation site(s) on the function of the receptor can then be evaluated. For determining the structure of the responsible glycan(s), mass spectrometry analysis enables us to evaluate both the occupancy and glycan structure on each glycosylation site. By using these approaches, the function and the structure of a specific glycan can be determined. Determining glycan structure is especially important when lectin or lectin-like molecules are involved in the regulation of glycoproteins. However, it is nearly impossible to manipulate the glycan structure of a specific glycoprotein in a living cell, therefore, it is difficult to confirm the indispensability of certain glycan structures in a specific molecule. Collecting data related to the glycan function of a large number of membrane proteins might provide a clue for elucidating the roles of specific glycan structures. This review summarizes progress that has been made concerning the glycosylation of cell surface receptors. receptors are classified as type I transmembrane receptor tyrosine kinases, and consist of an N-terminal extracellular domain, a transmembrane domain, an intracellular tyrosine kinase domain, and a C-terminal tail (Fig. 1). The binding of a ligand to the extracellular domain induces a conformational change from a "tethered form (the inactive form)" to an "extended form (the active form)" in which the dimerization arm mediates homo or heterodimers [13]. The receptor dimerization induces the phosphorylation of the tyrosine residues in the C-terminal tail, which subsequently activates the downstream signaling such as the PI3K/Akt pathway or the Ras/Erk pathway.
ErbB receptors are highly glycosylated. EGFR, ErbB2, ErbB3, and ErbB4 contain 12, 8, 10, and 11 N-glycosylation sites, respectively, in their extracellular domains. Figure 1 indicates the N-glycosylation sites of ErbB receptors, and the alignment of glycosylation sites of ErbB receptors, and the results indicate that some glycosylation sites are common among the four ErbB receptors.

N-glycans of EGFR
EGFR is a ~ 170 kDa protein with 11 typical (N-X-S/T, where X is any amino acid except proline) and 4 atypical (N-X-C) N-glycosylation consensus sequences [14]. Figure 2 indicates the occupancy and glycan structure on each N-glycosylation site of endogenous EGFR in A431 human epidermoid carcinoma cells [15], recombinant EGFR, which is expressed in CL-1 human lung cancer cells [16], and recombinant soluble EGFR (sEGFR, the extracellular domain of EGFR) expressed in CHO-K1 cells [17]. In the case of sEGFR in CHO-K1 cells, it was observed that all 11 typical N-glycosylation consensus sequences (N104, N151, N172, N328, N337, N389, N420, N504, N544, N579, N599) are either fully or partially glycosylated, and one of the four atypical N-glycosylation consensus sequences (N32) is fully glycosylated. It should also be noted that the occupancy and glycan structures are well conserved in all cell types that have been examined to date. Tyrosine kinase domain C-terminal tail  IV  III  II  I   C-terminal tail  IV  III  II  I   Tyrosine kinase domain C-terminal tail  IV  III  II  I   N599  N32   domain   I  I I  III  The N-glycosylation of EGFR is required for ligand binding [18,19], and the processing of oligosaccharides from high-mannose type to complex type does not affect this ability [20]. A site specific study has demonstrated that the N-glycan on N420 of EGFR is involved in dimerization [21]; among the four N-glycan deletion mutants of EGFR in which the glycosylation sites in domain III are mutated, the EGFR N420Q mutant exhibited ligand-independent oligomerization and phosphorylation. Another study reported that the deletion of the N-glycan on N579, which is in the domain IV auto-inhibitory tether loop, weakens the interaction between domains II and IV, thus increasing the ratio of high affinity binding receptors and also increasing the extent of ligand-independent dimerization [22]. These investigators assumed that N-glycan attached to N579 contributes to the stability of the inactive form, and that the deletion of the N-glycan might increase the structural flexibility of the moleculs. These studies suggest that specific N-glycans might be involved in stabilizing structure of EGFR to prevent unnecessary activation. Molecular dynamics simulation studies have also demonstrated that N-glycans are determinants of EGFR conformation, including the orientation of the extracellular domain relative to the membrane [23].
Glycan structures are also important for regulating EGFR when lectins or lectin-like molecules are involved. For example, in a previous study, we reported that the pulmonary surfactant protein D (SP-D) downregulates EGF signaling in lung adenocarcinoma [17,24]. SP-D is an apoprotein of  Fig. 2, EGFR contains high-mannose type N-glycans at N328 and N337, and SP-D possibly binds to these glycans. It is speculated that the binding of SP-D to EGFR directly interferes with EGF binding, or that SP-D affects the conformation of EGFR, thus altering the ligand binding characteristics of the molecule. It has been reported that EGFR with poly-N-acetyllactosamine (poly-LacNAc) containing N-glycans, which is produced by the enzymatic activity of N-acetylglucosaminyltransferase V (GnT-V), avoids constitutive endocytosis [27]. It has been proposed that galectin-3 binds to poly-LacNAc on the glycans of EGFR to form a molecular lattice, leading to the cell surface expression of EGFR being sustained. Intriguingly, it has been observed that the endocytosis of EGFR is increased in N-acetylglucosaminyltransferase III (GnT-III) transfected HeLaS3 cells [28]. GnT-III catalyzes the introduction of a GlcNAc unit to produce a "bisecting GlcNAc" structure [29], which prevents the formation of poly-LacNAc. It is possible that the binding of N-glycans of EGFR to galectin-3 is decreased by the activity of GnT-III, resulting in the upregulation of EGFR endocytosis. A case can be made that specific structures of N-glycans may regulate EGFR endocytosis through interactions with galectin-3.
The α1,6-fucosylation of N-glycans by the activity of α1,6 fucosyltransferase (Fut8) has been shown to affect EGFR function [30]. Fut8 catalyzes the addition of a fucose unit to the innermost GlcNAc residue of N-glycans, to produce α1,6-fucosylation, or a "core fucose" [31]. We previously reported that the loss of α1,6-fucosylation of N-glycans of EGFR reduces the binding of EGF to EGFR, and subsequent downstream signaling [30]. It has also been suggested that the increased sialylation and α1,3 fucosylation of the N-glycans of EGFR suppress EGF-induced EGFR dimerization [16,32].

N-glycans of ErbB3
ErbB3 is a receptor for neuregulin1 (heregulin), neuregulin 2, and neuregulin 6. The unique character of ErbB3 is its lack of tyrosine kinase activity [50]. Therefore, ErbB3 forms a heterodimer with other ErbB receptors and exerts downstream signaling, such as the PI3K/Akt pathway or Ras/Erk pathway. The activation of PI3K/Akt signaling has been implicated in the ErbB3-dependent progression of various types of cancer [51][52][53]. The induction of ErbB3 expression or signaling is an important factor in drug resistance in several cancer models. Because of the above findings, ErbB3 is considered to be a promising target for cancer therapy [54,55].
ErbB3 is a ~ 185 kDa protein with 10 N-glycosylation consensus sequences in the extracellular domain. Figure 3 provides a summary of the occupancy and glycan structure on each N-glycosylation site of recombinant soluble ErbB3 that is expressed in CHO-K1 cells. The N-glycan on N418 of ErbB3, which corresponds to N420 of EGFR based on sequence alignment (Fig. 1), is involved in dimer formation. Among the 10 single N-glycan deletion mutants of ErbB3, the ErbB3 N418Q mutant forms a heterodimer with ErbB2 without ligand stimulation, exerts downstream signaling and promotes tumor formation in athymic mice [56]. These findings suggest that the N-glycans play a role in maintaining the inactive form of ErbB receptors in the absence of a ligand. It is possible that the conformational changes from the inactive form to the active form in the N-glycan deletion mutant of ErbB3 N418Q requires less energy [57]. The extracellular domain of ErbB3 (= soluble ErbB3, sErbB3) exerts suppressive effects on heregulin signaling, and these effects are enhanced in N418Q mutant [58,59]. It is possible that the frequency of binding of the sErbB3 N418Q mutant to ErbB2 or other receptors on the cell surface is higher than that of the wild type [10].

EMT/MET Programs and glycans of cell surface receptor
We previously reviewed the role of N-glycosylation of several cell surface receptors such as TGF-β receptor, RTKs, Integrins, Wnt, Hedgehog, Notch, and the involvement of glycans in inflammation and hypoxia-induced EMT (endothelial-mesenchymal transition) in various kinds of diseases [9,60]. It is well known that phenotypic changes due to EMT play pivotal roles during embryonic development [61][62][63][64], wound healing [65], cancer and fibrosis [66]. Especially in cancer, invasion, metastasis and chemoresistance are considered to be highly associated with EMT [67]. The significance of EMT was also reported in COPD (chronic obstructive pulmonary disease) [68,69], interstitial pneumonitis [70], and lung cancer [69]. Moreover, EMT has also been implicated in resistance to therapy in cancer [67,71]. It has also been reported that EMT contributes to the development of resistance to the EGFR-tyrosine kinase inhibitor in non-small cell lung cancer [72]. The implication of COVID-19 infection has been also reported [73].
It was recently reported that an intermediate type of cell, referred to as a hybrid cell is produced between EMT and its reversive process, MET (mesenchymal-epithelial transition) [74][75][76]. A classical type of EMT programs were reported to be rather one-way processes, but recently it has become recognized that the EMT programs are more dynamic and can sometimes be reversed by epigenetic modification and gene regulation. There are distinct biomarkers for EMT and MET [77]. Among them, E-cadherin, claudin, occludin, and cytokeratin are typical examples because the gene expression patterns of these molecules are activated in MET and downregulated in EMT. On the other hand, N-cadherin, collagen, matrix metalloproteinases, fibronectin and vimentin are highly upregulated in EMT and downregulated in MET. There are also EMT transcription factors such as the Snail family referred to as SNAIL(SNAI1), SLUG (SNAI2) and SMUG (SNAI3), the basic helix-loop-helix protein family referred to as TWIST1 and TWIST2, and zing finger E-box binding transcription factors, referred to as ZEB1 and ZEB2 [67]. However, the issue of biomarkers for hybrid cells remains unclear at this time.
During EMT programs, the most important hallmark is TGF-β activation in various cancers. At the early stage of cancer, TGF-β acts as a protective factor whereas during carcinogenesis TGF-β functions as a progressive factor [78]. TGF-β is essential in developmental period as well. For example, TGF-β signaling facilitates the embryonic development of the lung and the aberrant glycosylation of TGF-β decreases signaling and downregulates the phosphorylation of Smad, thus causing emphysematous changes [79,80].
EGFR stimulates EMT during differentiation processes and apoptosis of cancer cells [81][82][83]. Resistance to therapy due to EGFR signaling has been reported [81]. Involvement of glycosphingolipids in EMT has also been demonstrated [84]. From these facts, changes in glycans in cell surface receptors may also play important roles in EMT and MET processes. Our group reported on the significance of branched N-glycans in various diseases, and in the case of EMT, our findings indicate that some glycosyltransferase genes of N-glycan branching and extension such as GnT-V, Fut8 and ST6Gal1 are upregulated in case of MET, whereas GnT-III is downregulated [85,86] as shown in Fig. 4. However, the expressions of those genes may be sometimes upregulated in both EMT and MET, and probably at the intermediate stage, i.e. in the hybrid cells, some of these enzyme expressions are reversible [60,87,88]. Response to reduction-oxidation (redox) is one of the most important biological phenomena for maintaining homeostasis of the body under conditions of various types of oxidative stress [89] under pathophysiological conditions. Our group has been interested in redox regulation under conditions of oxidative stress as well as glycobiology in relation to disease [90][91][92][93][94]. Functional and structural changes in glycans are regulated by redox responses resulting from the generation of reactive oxygen species (ROS) or reactive nitrogen species (RNS) in various diseases including cancer [95], diabetes [96], neurodegenerative diseases [97], such as Parkinson's disease, Alzheimer's disease as well as amyotrophic lateral sclerosis (ALS) (87), COPD [98] and aging. We proposed that the field "glyco-redox" investigations will open avenues to developing a more comprehensive understanding of the mechanism associated with diseases, as related to changes in glycan structures under oxidative stress [90]. The significance of this interplay was also reported by other groupes [99,100]. It is well known that signaling molecules such as PI3K/Akt [101], PKC-delta [102], Nrf2 [103], HIF1 [104] and Smad [105] are closely associated with redox regulation. Some of these signaling molecules also regulate the activities of glycosyltransferases that are involved in N-glycan biosynthesis such as ST6Gal1 [106], GnT-V and Fut8, etc.. It has also been reported that some transcription factors that regulate GnT-III, V, and Fut8 as well as ST6Gal1 may play key roles in the intermediate states. In order to regulate the EMT programs for preventing cancer development, progression, metastasis and therapy resistance, specific antibodies or specific inhibitors against various transcription factors toward N-glycan glycosyltransferases are likely candidates for novel therapeutics in the future as shown in Fig. 4.
Acknowledgements This work has been supported by JSPS KAK-ENHI Grant Numbers 21K06083 for MT and 18K06914 for NT, and by Japan Agency for Medical Research and Development (AMED) (20ac011070h005). We apologize for not citing all relevant papers due to space limitations. We wish to acknowledge Dr. Milton Feather for his English editing.

Data availability statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.