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

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, \u3b12,6-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

Glycosyltransferases in Cancer: Prognostic Biomarkers of Survival in Patient Cohorts and Impact on Malignancy in Experimental Models

Background: Glycosylation changes are a main feature of cancer. Some carbohydrate epitopes and expression levels of glycosyltransferases have been used or proposed as prognostic markers, while many experimental works have investigated the role of glycosyltransferases in malignancy. Using the transcriptomic data of the 21 TCGA cohorts, we correlated the expression level of 114 glycosyltransferases with the overall survival of patients. Methods: Using the Oncolnc website, we determined the Kaplan–Meier survival curves for the patients falling in the 15% upper or lower percentile of mRNA expression of each glycosyltransferase. Results: Seventeen glycosyltransferases involved in initial steps of N-or O-glycosylation and of glycolipid biosynthesis, in chain extension and sialylation were unequivocally associated with bad prognosis in a majority of cohorts. Four glycosyltransferases were associated with good prognosis. Other glycosyltransferases displayed an extremely high predictive value in only one or a few cohorts. The top were GALNT3, ALG6 and B3GNT7, which displayed a p < 1 × 10−9 in the low-grade glioma (LGG) cohort. Comparison with published experimental data points to ALG3, GALNT2, B4GALNT1, POFUT1, B4GALT5, B3GNT5 and ST3GAL2 as the most consistently malignancy-associated enzymes. Conclusions: We identified several cancer-associated glycosyltransferases as potential prognostic markers and therapeutic targets

The expanding roles of the Sda/Cad carbohydrate antigen and its cognate glycosyltransferase B4GALNT2

Background The histo-blood group antigens are carbohydrate structures present in tissues and body fluids, which contribute to the definition of the individual immunophenotype. One of these, the Sda antigen, is expressed on the surface of erythrocytes and in secretions of the vast majority of the Caucasians and other ethnic groups. Scope of review We describe the multiple and unsuspected aspects of the biology of the Sda antigen and its biosynthetic enzyme \u3b21,4-N-acetylgalactosaminyltransferase 2 (B4GALNT2) in various physiological and pathological settings. Major conclusions The immunodominant sugar of the Sda antigen is a \u3b21,4-linked N-acetylgalactosamine (GalNAc). Its cognate glycosyltransferase B4GALNT2 displays a restricted pattern of tissue expression, is regulated by unknown mechanisms - including promoter methylation, and encodes at least two different proteins, one of which with an unconventionally long cytoplasmic portion. In different settings, the Sda antigen plays multiple and unsuspected roles. 1) In colon cancer, its dramatic down-regulation plays a potential role in the overexpression of sialyl Lewis antigens, increasing metastasis formation. 2) It is involved in the lytic function of murine cytotoxic T lymphocytes. 3) It prevents the development of muscular dystrophy in various dystrophic murine models, when overexpressed in muscular fibers. 4) It regulates the circulating half-life of the von Willebrand factor (vWf), determining the onset of a bleeding disorder in a murine model. General significance The expression of the Sda antigen has a wide impact on the physiology and the pathology of different biological systems

High Expression of the Sda Synthase B4GALNT2 Associates with Good Prognosis and Attenuates Stemness in Colon Cancer

BACKGROUND: The carbohydrate antigen Sda and its biosynthetic enzyme B4GALNT2 are highly expressed in normal colonic mucosa but are down-regulated to a variable degree in colon cancer tissues. Here, we investigated the clinical and biological importance of B4GALNT2 in colon cancer. METHODS: Correlations of B4GALNT2 mRNA with clinical data were obtained from The Cancer Genome Atlas (TCGA) database; the phenotypic and transcriptomic changes induced by B4GALNT2 were studied in LS174T cells transfected with B4GALNT2 cDNA. RESULTS: TCGA data indicate that patients with high B4GALNT2 expression in cancer tissues display longer survival than non-expressers. In LS174T cells, expression of B4GALNT2 did not affect the ability to heal a scratch wound or to form colonies in standard growth conditions but markedly reduced the growth in soft agar, the tridimensional (3D) growth as spheroids, and the number of cancer stem cells, indicating a specific effect of B4GALNT2 on the growth in poor adherence and stemness. On the transcriptome, B4GALNT2 induced the down-regulation of the stemness-associated gene SOX2 and modulated gene expression towards an attenuation of the cancer phenotype. CONCLUSIONS: The level of B4GALNT2 can be proposed as a marker to identify higher- and lower-risk colorectal cancer patients

Biosynthesis and oligosaccharide structure of human CD8 glycoprotein expressed in a rat epithelial cell line.

The biosynthesis, post-translational modifications, and oligosaccharide structure of human CD8 glycoprotein have been studied in transfected rat epithelial cells. These cells synthesized and expressed on the plasma membrane high amounts of CD8 in a homodimeric form stabilized by a disulfide bridge. Three different CD8 forms were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after metabolic labeling and immunoprecipitation: a newly synthesized, unglycosylated 27-kDa (CD8u), a palmitylated and initially O-glycosylated 29-kDa (CD8i), and the mature, terminally glycosylated 32-34-kDa doublet (CD8m). CD8i is a transient intermediate form between CD8u and CD8m: characterization of carbohydrate moiety of [3H]glucosamine-labeled CD8i showed that it comprises for the vast majority non-elongated O-linked GalNAc closely spaced on the peptide backbone. Structural analysis of oligosaccharides released by mild alkaline borohydride treatment from the [3H]glucosamine-labeled CD8 34-kDa form showed that the neutral tetrasaccharide Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAcOH, and an homologous monosialylated pentasaccharide, predominate; the disialylated NeuAc2,3Gal beta 1,3(NeuAc alpha 2,6) GalNAcOH tetrasaccharide appeared to be poorly present. In the CD8 32-kDa form the neutral tetrasaccharide was by far the prominent O-linked chain, and no disialyloligosaccharides were identified. These results indicate that the maturation of CD8 glycoprotein in transfected rat epithelial cells results in the formation of branched O-linked oligosaccharides and that a higher degree of sialylation is responsible for the production of the heavier 34-kDa form

Impact of sialyltransferase ST6GAL1 overexpression on different colon cancer cell types

Cancer-associated glycan structures can be both tumor markers and engines of disease progression. The structure Sia\u3b12,6Gal\u3b21,4GlcNAc (Sia6LacNAc), synthesized by sialyltransferase ST6GAL1, is a cancer-associated glycan. Although ST6GAL1/Sia6LacNAc are often overexpressed in colorectal cancer (CRC), their biological and clinical significance remains unclear. To get insights into the clinical relevance of ST6GAL1 expression in CRC, we interrogated The Cancer Genome Atlas with mRNA expression data of hundreds of clinically characterized CRC and normal samples. We found an association of low ST6GAL1 expression with microsatellite instability (MSI), BRAF mutations and mucinous phenotype but not with stage, response to therapy and survival. To investigate the impact of ST6GAL1 expression in experimental systems, we analyzed the transcriptome and the phenotype of the CRC cell lines SW948 and SW48 after retroviral transduction with ST6GAL1 cDNA. The two cell lines display the two main pathways of CRC transformation: chromosomal instability and MSI, respectively. Constitutive ST6GAL1 expression induced much deeper transcriptomic changes in SW948 than in SW48 and affected different genes in the two cell lines. ST6GAL1 expression affected differentially the tyrosine phosphorylation induced by hepatocyte growth factor, the ability to grow in soft agar, to heal a scratch wound and to invade Matrigel in the two cell lines. These results indicate that the altered expression of a cancer-associated glycosyltransferase impacts the gene expression profile, as well as the phenotype, although in a cancer subtype-specific manner

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

<p>Abstract</p> <p>Background</p> <p>The 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.</p> <p>Methods</p> <p>Matched samples of urothelium and tumor tissue, and four bladder cancer cell lines were screened for: <it>ST3Gal.I</it>, <it>ST3Gal.II </it>and <it>ST3Gal.IV </it>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.</p> <p>Results</p> <p>In nonmuscle-invasive bladder cancers, <it>ST3Gal.I </it>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, <it>ST3Gal.I </it>mRNA levels were as elevated as nonmuscle-invasive cancers. Both non-malignant bladder tumors and corresponding urothelium showed <it>ST3Gal.I </it>mRNA levels lower than all the other specimen groups. A good correlation was observed in bladder cancer cell lines between the <it>ST3Gal.I </it>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 <it>ST3Gal.I </it>transcript variants were also equally expressed, independently of cell phenotype or malignancy.</p> <p>Conclusion</p> <p>ST3Gal.I plays the major role in the sialylation of the T antigen in bladder cancer. The overexpression of <it>ST3Gal.I </it>seems to be part of the initial oncogenic transformation of bladder and can be considered when predicting cancer progression and recurrence.</p

Differential Carbohydrate Recognition by Campylobacter jejuni Strain 11168: Influences of Temperature and Growth Conditions

The pathogenic clinical strain NCTC11168 was the first Campylobacter jejuni strain to be sequenced and has been a widely used laboratory model for studying C. jejuni pathogenesis. However, continuous passaging of C. jejuni NCTC11168 has been shown to dramatically affect its colonisation potential. Glycan array analysis was performed on C. jejuni NCTC11168 using the frequently passaged, non-colonising, genome sequenced (11168-GS) and the infrequently passaged, original, virulent (11168-O) isolates grown or maintained under various conditions. Glycan structures recognised and bound by C. jejuni included terminal mannose, N-acetylneuraminic acid, galactose and fucose. Significantly, it was found that only when challenged with normal oxygen at room temperature did 11168-O consistently bind to sialic acid or terminal mannose structures, while 11168-GS bound these structures regardless of growth/maintenance conditions. Further, binding of un-capped galactose and fucosylated structures was significantly reduced when C. jejuni was maintained at 25°C under atmospheric oxygen conditions. These binding differences identified through glycan array analysis were confirmed by the ability of specific lectins to competitively inhibit the adherence of C. jejuni to a Caco-2 intestinal cell line. Our data suggests that the binding of mannose and/or N-acetylneuraminic acid may provide the initial interactions important for colonisation following environmental exposure

Human Alpha Defensin 5 Expression in the Human Kidney and Urinary Tract

The mechanisms that maintain sterility in the urinary tract are incompletely understood. Recent studies have implicated the importance of antimicrobial peptides (AMP) in protecting the urinary tract from infection. Here, we characterize the expression and relevance of the AMP human alpha-defensin 5 (HD5) in the human kidney and urinary tract in normal and infected subjects.Using RNA isolated from human kidney, ureter, and bladder tissue, we performed quantitative real-time PCR to show that DEFA5, the gene encoding HD5, is constitutively expressed throughout the urinary tract. With pyelonephritis, DEFA5 expression significantly increased in the kidney. Using immunoblot analysis, HD5 production also increased with pyelonephritis. Immunostaining localized HD5 to the urothelium of the bladder and ureter. In the kidney, HD5 was primarily produced in the distal nephron and collecting tubules. Using immunoblot and ELISA assays, HD5 was not routinely detected in non-infected human urine samples while mean urinary HD5 production increased with E.coli urinary tract infection.DEFA5 is expressed throughout the urinary tract in non-infected subjects. Specifically, HD5 is expressed throughout the urothelium of the lower urinary tract and in the collecting tubules of the kidney. With infection, HD5 expression increases in the kidney and levels become detectable in the urine. To our knowledge, our findings represent the first to quantitate HD5 expression and production in the human kidney. Moreover, this is the first report to detect the presence of HD5 in infected urine samples. Our results suggest that HD5 may have an important role in maintaining urinary tract sterility