Distinct substrate specificities of human GlcNAc-6-sulfotransferases revealed by mass spectrometry-based sulfoglycomic analysis.

Sulfated glycans are known to be involved in several glycan-mediated cell adhesion and recognition pathways. Our mRNA transcript analyses on the genes involved in synthesizing GlcNAc-6-O-sulfated glycans in human colon cancer tissues indicated that GlcNAc6ST-2 (CHST4) is preferentially expressed in cancer cells compared with nonmalignant epithelial cells among the three known major GlcNAc-6-O-sulfotransferases. On the contrary, GlcNAc6ST-3 (CHST5) was only expressed in nonmalignant epithelial cells, whereas GlcNAc6ST-1 (CHST2) was expressed equally in both cancerous and nonmalignant epithelial cells. These results suggest that 6-O-sulfated glycans that are synthesized only by GlcNAc6ST-2 may be highly colon cancer-specific, as supported by immunohistochemical staining of cancer cells using the MECA-79 antibody known to be relatively specific to the enzymatic reaction products of GlcNAc6ST-2. By more precise MS-based sulfoglycomic analyses, we sought to further infer the substrate specificities of GlcNAc6STs via a definitive mapping of various sulfo-glycotopes and O-glycan structures expressed in response to overexpression of transfected GlcNAc6STs in the SW480 colon cancer cell line. By detailed MS/MS sequencing, GlcNAc6ST-3 was shown to preferentially add sulfate onto core 2-based O-glycan structures, but it does not act on extended core 1 structures, whereas GlcNAc6ST-1 prefers core 2-based O-glycans to extended core 1 structures. In contrast, GlcNAc6ST-2 could efficiently add sulfate onto both extended core 1- and core 2-based O-glycans, leading to the production of unique sulfated extended core 1 structures such as R-GlcNAc(6-SO3-)ß1-3Galß1-4GlcNAc(6-SO3-)ß1-3Galß1-3GalNAcα, which are good candidates to be targeted as cancer-specific glycans.

Colonic epithelial cells express specific ligands for mucosal macrophage immunosuppressive receptors siglec-7 and -9.

Immune cells are known to express specific recognition molecules for cell surface glycans. However, mechanisms involved in glycan-mediated cell-cell interactions in mucosal immunity have largely been left unaccounted for. We found that several glycans preferentially expressed in nonmalignant colonic epithelial cells serve as ligands for sialic acid-binding Ig-like lectins (siglecs), the immunosuppressive carbohydrate-recognition receptors carried by immune cells. The siglec ligand glycans in normal colonic epithelial cells included disialyl Lewis(a), which was found to have binding activity to both siglec-7 and -9, and sialyl 6-sulfo Lewis(x), which exhibited significant binding to siglec-7. Expression of these siglec-7/-9 ligands was impaired upon carcinogenesis, and they were replaced by cancer-associated glycans sialyl Lewis(a) and sialyl Lewis(x), which have no siglec ligand activity. When we characterized immune cells expressing siglecs in colonic lamina propriae by flow cytometry and confocal microscopy, the majority of colonic stromal immune cells expressing siglec-7/-9 turned out to be resident macrophages characterized by low expression of CD14/CD89 and high expression of CD68/CD163. A minor subpopulation of CD8(+) T lymphocytes also expressed siglec-7/-9. Siglec-7/-9 ligation suppressed LPS-induced cyclooxygenase-2 expression and PGE(2) production by macrophages. These results suggest that normal glycans of epithelial cells exert a suppressive effect on cyclooxygenase-2 expression by resident macrophages, thus maintaining immunological homeostasis in colonic mucosal membranes. Our results also imply that loss of immunosuppressive glycans by impaired glycosylation during colonic carcinogenesis enhances inflammatory mediator production.

Epigenetic silencing of the sulfate transporter gene DTDST induces sialyl Lewisx expression and accelerates proliferation of colon cancer cells.

Colon cancer cells express the carbohydrate determinant sialyl Lewis(x), while they exhibit markedly decreased the expression of its sulfated derivative, sialyl 6-sulfo Lewis(x). In contrast, normal colonic epithelial cells strongly express sialyl 6-sulfo Lewis(x), but they virtually do not express sialyl Lewis(x). Impaired sulfation was therefore suggested to occur during the course of malignant transformation of colonic epithelial cells and was assumed to be responsible for the increased sialyl Lewis(x) expression in cancers. To elucidate the molecular biological background of the impaired sulfation in cancers, we studied the expression levels of mRNA for 6-O-sulfotransferase isoenzymes, PAPS synthases and transporters, and a cell membrane sulfate transporter, DTDST, in cancer tissues. The most striking decrease in cancer cells compared with nonmalignant epithelial cells was noted in the transcription of the DTDST gene (P = 0.0000014; n = 20). Most cultured colon cancer cells had a diminished DTDST transcription, which was restored when cultured with histone deacetylase inhibitors. Suppression of DTDST transcription under the control of a tet-off inducible promoter resulted in increased sialyl Lewis(x) expression and reduced sialyl 6-sulfo Lewis(x) expression. Unexpectedly, the growth rate of the cancer cells was markedly enhanced when transcription of DTDST was suppressed. These results show that the decrease in the transcription of the sulfate transporter gene is the major cause of decreased expression of sialyl 6-sulfo Lewis(x) and increased expression of sialyl Lewis(x) in colon cancers. The results also suggest that the diminished DTDST expression is closely related to enhanced proliferation of cancer cells.

Altered expression of glycan genes in cancers induced by epigenetic silencing and tumor hypoxia: clues in the ongoing search for new tumor markers.

The glycan molecules that preferentially appear in cancers are clinically utilized as serum tumor markers. The exact reason, however, why glycans are useful as tumor markers remain elusive. Here, we will summarize lessons learned from well-established cancer-associated glycans, and propose strategies to develop new cancer markers. Our recent results on cancer-associated glycans, sialyl Lewis A and sialyl Lewis X, indicated that the repressed transcription of some glycan genes by epigenetic silencing during early carcinogenesis, and the transcriptional induction of some other glycan genes by tumor hypoxia accompanying cancer progression at locally advanced stages, are two major factors determining cancer-associated glycan expression. Multiple genes are involved in glycan synthesis, and epigenetic silencing of a part of such genes leads to accumulation of glycans having truncated incomplete structures, which are readily detected by specific antibodies. Glycans are very unique and advantageous as marker molecules because they are capable of reflecting epigenetic silencing in their structures. Transcriptional induction of some glycan genes by tumor hypoxia at the later stages produces further glycan modifications, such as an unusual increase of the N-glycolyl sialic acid residues in the glycan molecules. The entire process of malignant transformation thus creates abnormal glycans, whose structures reveal the effects of both epigenetic silencing and tumor hypoxia. The second advantage of a glycan marker over a proteinous marker is that they can reflect the plurality of genetic anomalies in a singular molecule, as it is synthesized by the cooperative action of multiple genes. Glycans are sometimes covalently bound to well-known cancer-associated proteins, such as CD44v, and this eventually contributes to a high cancer specificity and functional relevancy in cancer progression.

Clinical application of functional glycoproteomics - dissection of glycotopes carried by soluble CD44 variants in sera of patients with cancers.

We provide here an example of clinical application of functional glycoproteomics for cancer diagnosis. Sialyl Lewis a and sialyl Lewis x glycotopes, which are the specific ligands for selectins, and variant forms of CD44, which are the adhesion molecules recognizing hyaluronate, are both implicated in cancer metastasis. The CD44 variants modified by the sialyl Lewis a and sialyl Lewis x glycotopes are expected to have dual functions, serving as ligands for vascular selectins, and simultaneously having binding activity to vascular bed hyaluronate, and are expected to figure heavily in cancer metastasis. We developed a heterogeneous sandwich assay system to detect soluble CD44v specifically modified by the cancer-associated sialyl Lewis a/x glycotopes, using the extracellular domain of CD44v cleaved by the metalloproteinase ADAM10 as standard molecules. We also developed the assay system for CD44v modified by normal epithelial glycotopes including disialyl Lewis a and sialyl 6-sulfo Lewis x. The results indicated that serum levels of soluble CD44v modified by cancer-associated glycotopes were frequently increased in patients with cancers, while those of CD44v modified by the nonmalignant glycotopes tended to be elevated in patients with benign disorders.

Current relevance of incomplete synthesis and neo-synthesis for cancer-associated alteration of carbohydrate determinants--Hakomori's concepts revisited.

Incomplete synthesis and neo-synthesis are two major concepts for cancer-associated alterations of cell surface carbohydrate determinants, formulated by Hakomori and collaborators almost 25 years ago. These concepts are still as relevant and useful as ever for cancer-associated alteration of carbohydrate determinants. Incomplete synthesis of carbohydrate determinants occurs through the epigenetic silencing of glycogenes through DNA methylation and/or histone modification in the early stage cancers. The natural selection of more malignant cancer cells occurs through acquisition of hypoxia resistance by constitutively activated hypoxia inducible factors (HIFs) in the advanced stages of cancers. HIFs induce transcription of several important glycogenes, and lead to neo-synthesis of carbohydrate determinants. For instance, expression of sialyl Lewis A/X is induced by epigenetic silencing of glycogenes in the early stages, and is further accelerated in the advanced stages by hypoxia-induced transcription of several glycogenes. Expression of GM2 ganglioside is induced in cancers by altered glycosyltransferase activities, and its N-glycolyl sialic acid content increases by hypoxia-induced transcription of a sialic acid transporter gene. N-glycolyl GM2 thus reflects two cancer-associated genetic abnormalities in a single determinant, and has high cancer specificity. Every carbohydrate determinant is synthesized through multiple steps, each of which is affected by cancer-associated genetic abnormality. Superiority of carbohydrate determinants as cancer-specific molecules over protein determinants is demonstrated in that a single carbohydrate determinant can reflect multiple cancer-associated genetic abnormalities.

Human B-lymphocytes express alpha2-6-sialylated 6-sulfo-N-acetyllactosamine serving as a preferred ligand for CD22/Siglec-2.

CD22/Siglec-2, an important inhibitory co-receptor on B-lymphocytes, is known to recognize alpha2-6-sialylated glycan as a specific ligand. Here we propose that the alpha2-6-sialylated and 6-GlcNAc-sulfated determinant serves as a preferred ligand for CD22 because the binding of a human B-cell line to CD22 was almost completely abrogated after incubating the cells with NaClO3, an inhibitor of cellular sulfate metabolism, and was also significantly inhibited by a newly generated monoclonal antibody specific to the alpha2-6-sialylated 6-sulfo-N-acetyllactosamine (LacNAc) determinant (KN343, murine IgM). The alpha2-6-sialylated 6-sulfo-LacNAc determinant defined by the antibody was significantly expressed on a majority of normal human peripheral B-lymphocytes as well as follicular B-lymphocytes in peripheral lymph nodes. The determinant was also expressed in endothelial cells of high endothelial venules of secondary lymphoid tissues, including lymph nodes, tonsils, and intestine-associated lymphoid tissues, more strongly than on B-lymphocytes, suggesting a role for CD22 in B-cell interaction with blood vessels and trafficking. These results indicate that the alpha2-6-sialylated 6-sulfo-LacNAc determinant serves as an endogenous ligand for human CD22 and suggest the possibility that 6-GlcNAc sulfation as well as alpha2-6-sialylation may regulate CD22/Siglec-2 functions in humans.

Hypoxic culture induces expression of sialin, a sialic acid transporter, and cancer-associated gangliosides containing non-human sialic acid on human cancer cells.

Tumor hypoxia figures heavily in malignant progression by altering the intracellular glucose metabolism and inducing angiogenic factor production, thus, selecting and expanding more aggressive cancer cell clones. Little is known, however, regarding hypoxia-induced antigenic changes in cancers. We investigated the expression of N-glycolyl sialic acid (NeuGc)-G(M2), a cancer-associated ganglioside containing non-human sialic acid, NeuGc, in human cancers. Cancer tissues prepared from patients with colon cancers frequently expressed NeuGc-G(M2), whereas it was virtually absent in nonmalignant colonic epithelia. Studies on cultured cancer cells indicated that the non-human sialic acid was incorporated from culture medium. Hypoxic culture markedly induced mRNA for a sialic acid transporter, sialin, and this accompanied enhanced incorporation of NeuGc as well as N-acetyl sialic acid. Transfection of cells with sialin gene conferred accelerated sialic acid transport and induced cell surface expression of NeuGc-G(M2). We propose that the preferential expression of NeuGc-G(M2) in cancers is closely associated with tumor hypoxia. Hypoxic culture of tumor cells induces expression of the sialic acid transporter, and enhances the incorporation of non-human sialic acid from the external milieu. A consequence of this is the acquisition of cancer-associated cell surface gangliosides, typically G(M2), containing non-human sialic acid (NeuGc), which is not endogenously synthesized through CMP-N-acetyl sialic acid hydroxylase because humans lack the gene for the synthetic enzyme. As hypoxia is associated with diminished response to radiotherapy and chemotherapy, NeuGc-G(M2) is a potential therapeutic target for hypoxic cancer cells.

Loss of disialyl Lewis(a), the ligand for lymphocyte inhibitory receptor sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7) associated with increased sialyl Lewis(a) expression on human colon cancers.

Expression of sialyl Lewis(a) is known to be increased in cancers of the digestive organs. The determinant serves as a ligand for E-selectin and mediates hematogenous metastasis of cancers. In contrast, disialyl Lewis(a), which has an extra sialic acid attached at the C6-position of penultimate GlcNAc in sialyl Lewis(a), is expressed preferentially on nonmalignant colonic epithelial cells, and its expression decreases significantly on malignant transformation. Introduction of the gene for an alpha2-->6 sialyl-transferase responsible for disialyl Lewis(a) synthesis to colon cancer cells resulted in a marked increase in disialyl Lewis(a) expression and corresponding decrease in sialyl Lewis(a) expression. This was accompanied by the complete loss of E-selectin binding activity of the cells. In contrast, the transfected cells acquired significant binding activity to sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7)/p75/adhesion inhibitory receptor molecule-1, an inhibitory receptor expressed on lymphoid cells. These results indicate that the transition of carbohydrate determinants from disialyl Lewis(a)-dominant status to sialyl Lewis(a)-dominant status on malignant transformation has a dual functional consequence: the loss of normal cell-cell recognition between mucosal epithelial cells and lymphoid cells on one hand and the gain of E-selectin binding activity on the other. The transcription of a gene encoding the alpha2-->6 sialyltransferase was markedly down-regulated in cancer cells compared with nonmalignant epithelial cells, which is in line with the decreased expression of disialyl Lewis(a) and increased expression of sialyl Lewis(a) in cancers. Treatment of cancer cells with butyrate or 5-azacytidine induced strongly disialyl Lewis(a) expression, suggesting that histone deacetylation and/or DNA methylation may be involved in the silencing of the gene in cancers.

N-acetylglucosamine 6-O-sulfotransferase-1 regulates expression of L-selectin ligands and lymphocyte homing.

Lymphocyte homing is initiated by the binding of L-selectin on lymphocytes to its ligands on high endothelial venules (HEV). Sialyl 6-sulfo Lewis X is a major capping group of L-selectin ligands. N-Acetylglucosamine (GlcNAc) 6-sulfation is essential for the ligand activity, and is catalyzed by GlcNAc 6-O-sulfotransferases (GlcNAc6STs) of which GlcNAc6ST-1 and GlcNAc6ST-2 are expressed in HEV. Here, we report that mice deficient in GlcNAc6ST-1 were impaired in the elaboration of sialyl 6-sulfo Lewis X in HEV and that an epitope of L-selectin ligands recognized by the MECA-79 anti-body was greatly reduced or abolished in the abluminal aspect of HEV. Lymphocyte homing to peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches was significantly reduced in GlcNAc6ST-1 null mice. These results demonstrate that GlcNAc6ST-1 is involved in lymphocyte homing in vivo, and indicate that GlcNAc6ST-1 and -2 play complementary roles. The importance of GlcNAc6ST-1 is particularly high-lighted by its involvement in lymphocyte homing to Peyer's patches where GlcNAc6ST-2 expression is undetectable.

Carbohydrate-mediated cell adhesion in cancer metastasis and angiogenesis.

Malignant transformation is associated with abnormal glycosylation, resulting in the synthesis and expression of altered carbohydrate determinants including sialyl Lewisa and sialyl Lewisx. The sialyl Lewisa and sialyl Lewisx determinants appear in the sera of patients with cancer, and are extensively utilized for serum diagnosis of cancers in Japan. Sialyl Lewisa and sialyl Lewisx are involved in selectin-mediated adhesion of cancer cells to vascular endothelium, and these determinants are thought to be closely associated with hematogenous metastasis of cancers. Recent progress in this area includes the following: 1. Substantial increases in solid clinical statistics that further confirm the contribution of these determinants in the progression of a wide variety of cancers; 2. Elucidation of the ligand specificity of the three family members of selectins and evaluation of the roles of these molecules in cancer cell adhesion; and 3. Advances in the study of the mechanism that leads to the enhanced expression of the sialyl Lewis(a/x) determinants in malignant cells. These recent results have confirmed that these determinants are not merely markers for cancers, but are functionally implicated in the malignant behavior of cancer cells. The results also suggested that the increase of these determinants in malignant cells is an inevitable consequence of the malignant transformation of cells. Considerable new knowledge has also been accumulated regarding the therapeutic implications for suppression of hematogenous metastasis targeting this cell adhesion system.