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Sialyl Lewis x (sLex) is a selectin ligand whose overexpression in epithelial cancers mediates metastasis formation. The molecular basis of sLex biosynthesis in colon cancer tissues is still unclear. The prerequisite for therapeutic approaches aimed at sLex down-regulation in cancer, is the identification of rate-limiting steps in its biosynthesis. We have studied the role of α1,3-fucosyltransferases (Fuc-Ts) potentially involved in sLex biosynthesis in specimens of normal and cancer colon as well as in experimental systems. We found that: (i) in colon cancer, but not in normal mucosa where the antigen was poorly expressed, sLex correlated with a Fuc-T which, like Fuc-TVI, was active on 3′sialyllactosamine at a low concentration (Fuc-T SLN ); (ii) competitive RT-PCR analysis revealed that the level of Fuc-T mRNA expression in both normal and cancer colon was Fuc-TVI > Fuc-TIII > Fuc-TIV; Fuc-TV and Fuc-TVII expression was negligible; (iii) sLex was expressed only by the gastrointestinal cell lines displaying both Fuc-TVI mRNA and Fuc-T SLN activity, but not by those expressing only Fuc-TIII mRNA; (iv) transfection with Fuc-TVI cDNA, but not with Fuc-TIII cDNA, induced sLex expression in gastrointestinal cell lines; (v) Fuc-TVI knock-down with specific siRNA induced down-regulation of Fuc-TVI mRNA and Fuc-T SLN activity and a dramatic inhibition of sLex expression. These data indicate that in colon cancer tissues Fuc-TVI is a key regulator of sLex biosynthesis which can be the target of RNA-interference-based gene knock-down approaches.

Background Glycosylation is increasingly recognized as one of the most relevant postranslational modifications. Sialic acids are negatively charged sugars which frequently terminate the carbohydrate chains of glycoproteins and glycolipids. The addition of sialic acids is mediated by sialyltransferases, a family of glycosyltransferases with a crucial role in cancer progression. Scope of the review To describe the phenotypic and clinical implications of altered expression of sialyltransferases and of their cognate sialylated structures in cancer. To propose a unifying model of the role of sialyltransferases and sialylated structures on cancer progression. Major conclusions We first discuss the biosynthesis and the role played by the major cancer-associated sialylated structures, including Thomsen–Friedenreich-associated antigens, sialyl Lewis antigens, α2,6-sialylated lactosamine, polysialic acid and gangliosides. Then, we show that altered sialyltransferase expression in cancer, consequence of genetic and epigenetic alterations, generates a flow of information toward the membrane through the biosynthesis of aberrantly sialylated molecules (inside-out signaling). In turn, the presence of aberrantly sialylated structures on cell membrane receptors generates a flow of information toward the nucleus, which can exacerbate the neoplastic phenotype (outside-in signaling). We provide examples of self-fueling loops generated by these flows of information. General significance Sialyltransferases have a wide impact on the biology of cancer and can be the target of innovative therapies. Our unified view provides a conceptual framework to understand the impact of altered glycosylation in cancer.

Apoptosis profoundly alters the carbohydrate layer coating the membrane of eukaryotic cells. Previously we showed that apoptotic cells became reactive with the alpha2, 6-sialyl-specific lectin from Sambucus nigra (SNA), regardless of their histological origin and the nature of the apoptotic stimulus. Here we reveal the basis of the phenomenon by showing that in apoptotic cancer cell lines SNA reactivity was mainly associated with a 67 kDa glycoprotein which we identified by MALDI-TOF/TOF and immunoblot analysis as bovine vitronectin (bVN). bVN was neither present in non-apoptotic cells, nor in cells induced to apoptosis in serum-free medium, indicating that its uptake from the cell culture serum occurred only during apoptosis. The bVN molecules associated with apoptotic cancer cell lines represented minor isoforms, lacking the carboxyterminal sequence and paradoxically containing a few alpha2,6-linked sialic acid residues. Despite their poor alpha2,6-sialylation, these bVN molecules were sufficient to turn apoptotic cells to SNA reactivity, which is a late apoptotic event occurring in cells positive to both annexin-V and propidium iodide. Unlike in cancer cell lines, the major bVN form taken up by apoptotic neutrophils and mononuclear cells was a 80 kDa form. In apoptotic SW948 cells we also detected the alpha2,6-sialylated forms of the stress-70 mitochondrial precursor (mortalin) and of tubulin-beta2C. These data indicate that the acquisition of vitronectin isoforms from the environment is a general, although cell specific phenomenon, potentially playing an important role in post-apoptotic events and that the alpha2,6- sialylation of intracellular proteins is a new kind of posttranslational modification associated with apoptosis.

The process of cell transformation induces profound alterations in the glycosylation pattern of the cells. Often, the carbohydrate structures formed by cancer cells resemble those expressed by the corresponding normal tissue during the fetal life (onco-developmental regulation) and are largely tissue-specific. However, some structures appear to be widely expressed by cancers of different histological origin. This is probably due to two main reasons: first, their biosynthesis is strictly controlled by the mechanisms altered in cell transformation (i.e. activation of oncogenes, inactivation of tumour-suppressor genes, altered pattern of epigenetic regulation); second, their expression provide cancer cells with a growth advantage, resulting in the selection of cells expressing a given antigen during tumour growth. In the following sections, we will first describe some of the most widely expressed cancer-related carbohydrate structures (Section 2), then we will discuss how the altered mechanisms controlling cell growth in cancer influence the glycosylation machinery (Section 3). Last, we will discuss how the cancer-related carbohydrate structures modify the cancer cell phenotype (Section 4).

Sialyl Lewis x (sLex) is a well known selectin ligand involved in metastasis. The molecular bases of its overexpression in colon cancer are still unclear. Here we show that FucT-VI is the main fucosyltransferase responsible for sLex biosynthesis in colonic tissues and a major regulator of sLex expression in colon cancer because: 1) in colon cancer specimens and cell lines sLex expression correlates with the activity of a fucosyltransferase able to fucosylate 3’ sialyllactosamine (3’SLN) at a low (0.5 mM) acceptor concentration. 2) In cells transiently transfected with FucT genes only FucT-VI displays this property. 3) In normal and cancer colon, FucT-VI transcript shows the highest level of expression (about 200 fg/pg actin, vs. 30 for FucT-III, 10 for FucT-IV

Many observations have reported glycosylation changes associated with apoptosis in different biological systems, although none of these has shown any general significance. In this work, we show that in cell lines from different histological origin, (colon, breast, pancreas, and bladder cancer) as well as in normal human and mice neutrophils, apoptosis is accompanied by the exposure of sugar chains recognized by the lectin from Sambucus nigra (SNA), specific for Sia alpha 2,6Gal/GalNAc structures. Also, cells undergoing primary necrosis induced by heat treatment (56 degrees C, 30 min) expose specifically binding sites for SNA. While this modification is recognized also by the lectin from the mushroom Polyporus squamosus, which is highly specific for alpha2,6-sialylated lactosamine, no significant changes were detected in the binding of lectins specific for other carbohydrate structures, such as those from Phaseolus vulgaris, Arachis hypogea, and Maackia amurensis. The binding of SNA to apoptotic/necrotic cells is inhibited by neuraminidase treatment and by alpha2,6-sialylated compounds. In apoptotic, but not in necrotic SW948 cells, SNA reactivity is specifically associated with 65, 69, and 87 kDa glycoproteins. The exposure of SNA-reactive chains by apoptotic/necrotic cells occurs also in cells not expressing sialyltransferases ST6Gal.1 or ST6Gal.2 and is largely independent of the presence of alpha2,6-sialylated lactosaminic chains on the surface of preapoptotic cells. In neutrophils from ST6Gal.1 knock-out mice, the apoptosis-related increase in SNA reactivity is reduced but not abolished. These data demonstrate that apoptosis and primary necrosis induce a specific glycosylation change independent of the cell type and nature of the stimulus.

The carbohydrate determinants Sd(a) and sialyl Lewis x (sLex) both result from substitution of an alpha2,3-sialylated type 2 chain: the first with an N-acetylgalactosamine (GalNAc) beta1,4-linked to Gal and the second by an alpha1,3-linked fucose on N-acetylglucosamine. The Sd(a) antigen is synthesized by Sd(a) beta1,4-N-acetylgalactosaminyltransferase II (beta4GalNAcT-II), which is downregulated in colon cancer, whereas sLex is a cancer-associated antigen. In view of the possible competition between beta4GalNAcT-II and the fucosyltransferases (FucTs) synthesizing the sLex antigen, we investigated whether beta4GalNAcT-II acts as a negative regulator of sLex expression in colon cancer. beta4GalNAcT-II cDNA, when expressed in LS174T colon cancer cells, induces the expression of the Sd(a) antigen, a dramatic inhibition of sLex expression on cell membranes, and the replacement of sLex with the Sd(a) antigen on 290 kDa glycoproteins. Unexpectedly, in colorectal cancer specimens, beta4GalNAcT-II and sLex show a direct relation. The reasons appear to be (i) Sd(a) and sLex antigens are expressed by different glycoproteins of 340 and 290 kDa, respectively; (ii) the activity of alpha1,3-FucTs on 3'-sialyllactosamine parallels that of beta4GalNAcT-II; and (iii) both beta4GalNAcT-II and FucT activities parallel sLex expression. Quantitative reverse transcription-polymerase chain reaction analysis reveals that the transcripts of beta4GalNAcT-II and those of FucT-III and FucT-VII are positively correlated. These data indicate that in colon cancer tissues, the sLex antigen is regulated mainly by the total FucT activity on 3'-sialyllactosamine acceptors and that beta4GalNAcT-II can inhibit sLex expression in an experimental model, although not in colon cancer tissues.

Beta-Galactoside alpha2,6 sialyltransferase (ST6Gal.I) is the principal sialyltransferase responsible for the biosynthesis of the sialyl alpha2,6-galactosyl linkage. This enzyme and its cognate glycosidic structure are overexpressed in several malignancies and are related to cancer progression. The expression of the enzyme is regulated mainly through the expression of three principal mRNA species differing in the 5' untranslated exons. The form known as YZ is considered associated with the basal expression of the gene, while forms H and X are specific of the liver and of B-lymphocytes, respectively. Using a panel of human cancer cell lines we have studied the expression of ST6Gal.I activity by two different methods, the expression of alpha2,6-sialylated sugar chains by the lectin from Sambucus nigra (SNA) and the expression of the different mRNA species by RT-PCR using oligonucleotide primers complementary to the isoform-specific regions. We report that very high levels of ST6Gal.I activity result in high levels of SNA reactivity and are associated with the expression of the H transcript in colon and liver cell lines and of the X transcript in B cells.

Cell membrane glycoconjugates undergo characteristic changes as a consequence of neoplastic transformation. The cancer-associated carbohydrate structures play key roles in cancer progression by altering the cell-cell and cell-environment interactions. In this review, we will discuss some of the most relevant cancer-associated carbohydrate structures, including the β1,6-branching of N-linked chains, the sialyl Lewis antigens, the α2,6-sialylated lactosamine, the Thomsen-Friedenreich-related antigens and gangliosides. We will describe the mechanisms leading to the expression of these structures and their interactions with sugar binding molecules, such as selectins and galectins. Finally, we will discuss how the glycosylation machinery of the cell is controlled by signal transduction pathways, epigenetic mechanisms and responds to hypoxia.