Fabio Dall'Olio

Sialyltransferases in cancer

It has long been known that cancer cells often express more heavily sialylated glycans on their surface and that this feature sometimes correlates with invasion. It is now well established that specific sialylated structures, such as the Thomsen-Friedenreich-related antigens, the sialyl Lewis antigens, the sialyl α2-6 lactosaminyl structure, the polysialic acid or some gangliosides, can mediate cellular interactions and are altered in cancer cells. This review summarizes the current knowledge on the cancer-associated alterations in sialyltransferase expression which are often at the basis of the deranged expression of sialylated structures.

Mechanisms of cancer-associated glycosylation changes.

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.

The sialyl-α2,6-lactosaminyl-structure: Biosynthesis and functional role

Sialylation represents one of the most frequently occurring terminations of the oligosaccharide chains of glycoproteins and glycolipids. Sialic acid is commonly found α,3- or α,6-linked to galactose (Gal), α,6-linked to N-acetylgalactosamine (GalNAc) or α,8-linked to another sialic acid. The biosynthesis of the various linkages is mediated by the different members of the sialyltransferase family. The addition of sialic acid in α,6-linkage to the galactose residue of lactosamine (type 2 chains) is catalyzed by β-galactoside α,6-sialyltransferase (ST6Gal.I). Although expressed by a single gene, this enzyme shows a complex pattern of regulation which allows its tissue- and stage-specific modulation. The cognate oligosaccharide structure, NeuAcα,6Galβ1,4GIcNAc, is widely distributed among tissues and is involved in biological processes such as the regulation of the immune response and the progression of colon cancer. This review summarizes the current knowledge on the biochemistry of ST6Gal.I and on the functional role of the sialyl-α,6-lactosaminyl structure.

Protein glycosylation in cancer biology: an overview

Introduction Glycosylation is one of the most frequentlyoccurring post-translational modifications of proteins. The presence of an oligosaccharide moiety in soluble and membrane bound proteins improves solubility in water, contributes to the proper orientation of the molecule, protects it from proteases and, in some cases, is required for efficient intracellular transport. Some oligosaccharide sequences may also mediate highly specific molecular and cellular recognition. In the first section of this review the biochemistry of protein glycosylation is summarised briefly; the second section deals with cancer related oligosaccharide changes and invasive properties of cancer cells, with special emphasis on experimental systems; the third section focuses on specific oligosaccharide structural alterations in human cancers.

ST3Gal.I sialyltransferase relevance in bladder cancer tissues and cell lines

BackgroundThe T antigen is a tumor-associated structure whose sialylated form (the sialyl-T antigen) involves the altered expression of sialyltransferases and has been related with worse prognosis. Since little or no information is available on this subject, we investigated the regulation of the sialyltransferases, able to sialylate the T antigen, in bladder cancer progression.MethodsMatched samples of urothelium and tumor tissue, and four bladder cancer cell lines were screened for: ST3Gal.I, ST3Gal.II and ST3Gal.IV mRNA level by real-time PCR. Sialyl-T antigen was detected by dot blot and flow cytometry using peanut lectin. Sialyltransferase activity was measured against the T antigen in the cell lines.ResultsIn nonmuscle-invasive bladder cancers, ST3Gal.I mRNA levels were significantly higher than corresponding urothelium (p < 0.001) and this increase was twice more pronounced in cancers with tendency for recurrence. In muscle-invasive cancers and matching urothelium, ST3Gal.I mRNA levels were as elevated as nonmuscle-invasive cancers. Both non-malignant bladder tumors and corresponding urothelium showed ST3Gal.I mRNA levels lower than all the other specimen groups. A good correlation was observed in bladder cancer cell lines between the ST3Gal.I mRNA level, the ST activity (r = 0.99; p = 0.001) and sialyl-T antigen expression, demonstrating that sialylation of T antigen is attributable to ST3Gal.I. The expression of sialyl-T antigens was found in patients bladder tumors and urothelium, although without a marked relationship with mRNA level. The two ST3Gal.I transcript variants were also equally expressed, independently of cell phenotype or malignancy.ConclusionST3Gal.I plays the major role in the sialylation of the T antigen in bladder cancer. The overexpression of ST3Gal.I seems to be part of the initial oncogenic transformation of bladder and can be considered when predicting cancer progression and recurrence.

Sialosignaling: sialyltransferases as engines of self-fueling loops in cancer progression.

BACKGROUNDnGlycosylation 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.nnnSCOPE OF THE REVIEWnTo 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.nnnMAJOR CONCLUSIONSnWe 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.nnnGENERAL SIGNIFICANCEnSialyltransferases 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.

β-Galactoside α2,6 sialyltransferase in human colon cancer : Contribution of multiple transcripts to regulation of enzyme activity and reactivity with Sambucus nigra agglutinin

Colon cancer tissues display an increased activity of β‐galactoside α2,6 sialyltransferase (ST6Gal.I) and an increased reactivity with the lectin from Sambucus nigra (SNA), specific for α2,6‐sialyl‐linkages. Experimental and clinical studies indicate a contribution of these alterations to tumor progression, but their molecular bases are largely unknown. In many tissues, ST6Gal.I is transcriptionally regulated through the usage of different promoters that originate mRNAs diverging in the 5`‐untranslated regions. RT‐PCR analysis of 14 carcinoma samples, all expressing an increased ST6Gal.I enzyme activity, and of the corresponding normal mucosa revealed the presence of at least 2 transcripts. One, containing the 5`‐untranslated exons, Y+Z, is thought to represent the “housekeeping” expression, and another previously described in hepatic tissues. Both the Y+Z and the hepatic transcripts were detectable in normal and cancer tissues but that latter form had a marked tendency to accumulate in cancer. The extent of α2,6‐sialylation of glycoconjugates, as determined by SNA‐dot blot analysis, was markedly enhanced in all cancer specimens, but the level of reactivity only partially correlated with the level of enzyme expression. Western blot analysis revealed a strikingly heterogeneous pattern of SNA reactivity among cancer tissues. These data indicate that: i) during neoplastic transformation of colonic cells, ST6Gal.I expression may be modulated through a differential promoter usage; ii) the extent of α2,6‐sialylation of cancer cell membranes is not a direct function of the ST6Gal.I activity, strongly suggesting the existence of other, more complex mechanisms of regulation. Int. J. Cancer 88:58–65, 2000.

High-mannose oligosaccharides from human Tamm-Horsfall glycoprotein

The present communication reports the occurrence of high-mannose oligosaccharides on Tamm-Horsfall glycoprotein prepared from human pooled urine. The Pronase digest of the glycoprotein was fractionated by gel filtration and a high-mannose glycopeptide species was separated from complex-type glycopeptides. When high-mannose glycopeptides were digested with endo-β-N-acetylglucosaminidase H, followed by reduction with [3H]KBH4, three oligosaccharides were resolved by thin-layer chromatography. On the basis of chromatographic mobility and exoglycosidase digestions the composition Man7-, Man6-, and Man5-GlcNAc was assigned to the three oligosaccharides. Man6GlcNAc is by far the major component.

Rapid isolation of Tamm-Horsfall glycoprotein (uromodulin) from human urine

An isolation method for Tamm-Horsfall protein is described which is based on the observation that a diatomaceous earth filter is able to retain most of the glycoprotein present in urine and that the glycoprotein is easily desorbed from the filter by deionized water. This behaviour depends on the tendency of Tamm-Horsfall glycoprotein at normal urinary concentrations to form a gel in a solution containing mono- and divalent ions. By means of two-step filtration, the glycoprotein was purified to homogeneity. The yield was of about 20 mg/l of urine, and the time required for the isolation was approximately 5-6 h. This procedure should be particularly useful for preparing large amounts of Tamm-Horsfall glycoprotein oligosaccharides in order to investigate their potential use as immunosuppressive agents both in vitro and in vivo.

Differential expression of the hepatic transcript of β-galactoside α2,6-sialyltransferase in human colon cancer cell lines

The activity of β‐galactoside α2,6‐sialyltransferase (ST6Gal.1), the enzyme responsible for the addition of sialic acid in α2,6‐linkage to N‐acetyllactosaminic (Galβ1,4GlcNAc) units of glycoconjugates, is increased in the vast majority of colon cancer specimens, and a positive correlation with an invasive phenotype has been suggested by several studies. In many tissues, ST6Gal.1 is regulated mainly at the transcriptional level through the use of different cell‐specific promoters which generate transcripts differing in their 5‐untranslated regions. With the aim of understanding the molecular bases of the increased ST6Gal.1 expression in colon cancer, we investigated the expression of mRNA species in colon cancer cell lines and the relationship with enzyme activity and extent of α2,6‐sialylation of cell glycoproteins. All cell lines examined express the form containing the 5‐untranslated exons Y and Z, typical of the “basal” expression of the gene, while others express also the liver transcript. This indicates that colon cancer cell lines can be grouped according to expression of the liver transcript of ST6Gal.1. The cell lines expressing only the Y+Z form display, in general, a lower activity:mRNA ratio, which might indicate reduced translational efficiency. The level of α2,6‐sialylation of cell glycoproteins, as determined by reactivity with the Sambucus nigra lectin, is closely associated with the level of enzyme activity. Int. J. Cancer 81:243–247, 1999.