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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.

Simple Summary The glycosyltransferase β1,4-N-acetylgalactosaminyltransferae 2 (B4GALNT2), product of the B4GALNT2 gene is responsible for the biosynthesis of the carbohydrate antigen Sda. Both the enzyme and its cognate antigen display a restricted pattern of tissue expression and modulation in colorectal, gastric, and mammary cancers. In colorectal cancer, B4GALNT2 is generally downregulated, but patients displaying higher expression survive longer. The sialyl Lewisa and sialyl Lewisx antigens are associated with malignancy. Their biosynthesis and that of Sda are mutually exclusive. Forced expression of B4GALNT2 in colorectal cancer cell lines modulates the transcriptome towards lower malignancy, reducing stemness. These effects are independent of B4GALNT2-induced sLea/sLex inhibition. Thus, B4GALNT2 is a marker of better prognosis and a cancer-restraining enzyme in colorectal cancer, with a therapeutic potential. Abstract Terminal carbohydrate structures are particularly relevant in oncology because they can serve as cancer markers and alter the phenotype of cancer cells. The Sda antigen and the sialyl Lewisx and sialyl Lewisa (sLex and sLea) antigens are terminal structures whose biosynthesis is mutually exclusive. In this review, we describe the main features of the Sda antigen in cancer and its relationship with sLex/a antigens. Information was obtained from an extensive literature search and from The Cancer Genome Atlas (TCGA) public database. The Sda biosynthetic enzyme B4GALNT2 undergoes downregulation in colorectal (CRC) and stomach cancer, while it is ectopically expressed by a minority of breast cancer (BRCA) patients. High expression of B4GALNT2 is associated with better prognosis and a less malignant gene expression profile in CRC, while the opposite occurs in BRCA. The regulation of B4GALNT2 expression in CRC is multifactorial, involving gene methylation and miRNA expression. Forced expression of B4GALNT2 inhibited sLea/sLex and reduced malignancy and stemness in cells constitutively expressing sLex/a antigens. However, consistent effects were observed upon B4GALNT2 forced expression and in cells not expressing sLex/a antigens. Thus, B4GALNT2 and the Sda antigen exert a tumor-restraining activity in CRC and probably other gastrointestinal cancers, independently of sLex/a antigens.

Background: glycosyltransferase B4GALNT2 and its cognate carbohydrate antigen Sda are highly expressed in normal colon but strongly downregulated in colorectal carcinoma (CRC). We previously showed that CRC patients expressing higher B4GALNT2 mRNA levels displayed longer survival. Forced B4GALNT2 expression reduced the malignancy and stemness of colon cancer cells. Methods: Kaplan–Meier survival curves were determined in “The Cancer Genome Atlas” (TCGA) COAD cohort for several glycosyltransferases, oncogenes, and tumor suppressor genes. Whole expression data of coding genes as well as miRNA and methylation data for B4GALNT2 were downloaded from TCGA. Results: the prognostic potential of B4GALNT2 was the best among the glycosyltransferases tested and better than that of many oncogenes and tumor suppressor genes, high B4GALNT2 expression was associated with a lower malignancy gene expression profile, differential methylation of an intronic B4GALNT2 gene position and miR-204-5p expression play major roles in B4GALNT2 regulation. Conclusions: high B4GALNT2 expression is a strong predictor of good prognosis in CRC as a part of a wider molecular signature that includes ZG16, ITLN1, BEST2, and GUCA2B. Differential DNA methylation and miRNA expression contribute to regulating B4GALNT2 expression during colorectal carcinogenesis.

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.

Glycosylation consists in the covalent, enzyme mediated, attachment of sugar chains to proteins and lipids. A large proportion of membrane and secreted proteins are indeed glycoproteins, while glycolipids are fundamental component of cell membranes. The biosynthesis of sugar chains is mediated by glycosyltransferases, whose level of expression represents a major factor of regulation of the glycosylation process. In cancer, glycosylation undergoes profound changes, which often contribute to invasion and metastasis. Epithelial to mesenchymal transition (EMT) is a key step in metastasis formation and is intimately associated with glycosylation changes. Numerous carbohydrate structures undergo up- or down-regulation during EMT and often regulate the process. In this review, we will discuss the relationship with EMT of the N-glycans, of the different types of O-glycans, including the classical mucin-type, O-GlcNAc, O-linked fucose, O-linked mannose and of glycolipids. Finally, we will discuss the role in EMT of galectins, a major class of mammalian galactoside-binding lectins. While the expression of specific carbohydrate structures can be used as a marker of EMT and of the propensity to migrate, the manipulation of the glycosylation machinery offers new perspectives for cancer treatment through inhibition of EMT.

Background: The Sda antigen and its biosynthetic enzyme B4GALNT2 are highly expressed in healthy colon but undergo a variable down-regulation in colon cancer. The biosynthesis of the malignancy-associated sialyl Lewis x (sLex) antigen in normal and cancerous colon is mediated by fucosyltransferase 6 (FUT6) and is mutually exclusive from that of Sda. It is thought that the reduced malignancy associated with high B4GALNT2 was due to sLex inhibition. Methods: We transfected the cell lines SW480 and SW620, derived respectively from a primary tumor and a metastasis of the same patient, with the cDNAs of FUT6 or B4GALNT2, generating cell variants expressing either the sLex or the Sda antigens. Transfectants were analyzed for growth in poor adherence, wound healing, stemness and gene expression profile. Results: B4GALNT2/Sda expression down-regulated all malignancy-associated phenotypes in SW620 but only those associated with stemness in SW480. FUT6/sLex enhanced some malignancy-associated phenotypes in SW620, but had little effect in SW480. The impact on the transcriptome was stronger for FUT6 than for B4GALNT2 and only partially overlapping between SW480 and SW620. Conclusions: B4GALNT2/Sda inhibits the stemness-associated malignant phenotype, independently of sLex inhibition. The impact of glycosyltransferases on the phenotype and the transcriptome is highly cell-line specific.

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.

Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced byB. longum. This has been valuedin vitro, performing the incubation of threeB. longum strains with enterocyte-like Caco-2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells withB. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherentB. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues.

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.

International audience; 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 Sd(a) 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 Sd(a) antigen and its biosynthetic enzyme β1,4-N-acetylgalactosaminyltransferase 2 (B4GALNT2) in various physiological and pathological settings.MAJOR CONCLUSIONS:The immunodominant sugar of the Sd(a) antigen is a β1,4-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 Sd(a) 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 Sd(a) antigen has a wide impact on the physiology and the pathology of different biological systems.

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).

Background 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. Methods Matched 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. Results In 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. Conclusion ST3Gal.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.

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.

Apoptosis is a tightly regulated process of cell death which results in cell fragmentation and removal of demised cells by phagocytosis. The several glycosylation changes described in apoptotic cells, usually by means of fluorescent lectins, are often cell specific and the identification of apoptosis-associated carbohydrate changes of general significance is still lacking. Sambucus nigra agglutinin (SNA) is a widely used tool for the detection of 2,6-sialylated lactosamine, a carbohydrate epitope often associated with cancer, which is the product of sialyltransferase ST6Gal.1. In this study, we show that cell lines of different histological origin, such as colon, breast, pancreas and bladder cancer undergoing apoptosis display a strong increase of SNA reactivity, through a ST6Gal.1-independent mechanism. This change is specific, in that the reactivity with other lectins, such as Maakia amurensis lectin, peanut agglutinin and leukoagglutinin does not show any consistent change. Surprisingly, also a heat treatment which induces primary necrosis causes a specific increase of SNA reactivity. In the colon cancer cell lines SW48 and SW948 which are basically devoid of measurable ST6Gal.1 activity and which express very low levels of SNA reactivity, apoptosis- or necrosis-inducing treatments induce increased SNA reactivity but do not activate the dormant ST6Gal.1 activity. The spontaneous apoptosis occurring in human neutrophiles at the end of their brief life, also causes a strong increase of SNA reactivity. Together, these data indicate that SNA detects specifically a change occurring in apoptotic and necrotic cells, regardless their histological origin and the nature of the apoptotic stimulus.

Sialyl Lewis X (sLex) is a well known carbohydrate antigen whose overexpression in cancer correlates with metastasis. In colon cancer, the molecular bases of sLex overexpression remain elusive. We have investigated sLex expression in normal and cancer colonic tissues and in cell lines as a function of the expression of fucosyltransferases and beta4GalNAcT-II. This enzyme, which is downregulated in colon cancers, is responsible for the biosynthesis of the Sda antigen [Siaalpha2,3(GalNAcbeta1,4)Galbeta1,4GlcNAc], whose biosynthesis competes with that of sLex. In colon cancers and cell lines, sLex expression correlates with a fucosyltransferase activity able to fucosylate 3'sialyllactosamine at low (0.5 mM) concentration. Transfection experiments with FucT-III, IV, V, VI and VII cDNAs in COS-7 cells indicate that only FucT-VI displays this property. In gastrointestinal cell lines, high levels of this fucosyltransferase activity are shown by cell lines expressing high levels of the FucT-VI transcript. In normal colon, sLex antigen is detectable upon de-acetylation but its expression does not correlate with any fucosyltransferase activity or transcript. Rather, we observed a significant correlation between sLex and the ratio between fucosyltransferase activity with 0.5 mM 3'sialyllactosamine and beta4GalNAcT-II activity. These data suggest that in both normal and cancer colonic tissues the biosynthesis of sLex is mainly due to FucT-VI or to a fucosyltransferase with similar kinetic properties, unknown at present. In normal colon, but not in colon cancers, the fucosyltransferase activity above described is counteracted by competing glycosyltransferases, as the high beta4GalNAcT-II activity that synthesizes the alternative Sda antigen.

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.

The histo-blood group carbohydrate determinant Sda is expressed by about 95% of the individuals of Caucasian origin. The addition of the immunodominant sugar, a beta1,4-linked N-acetylgalactosamine, is mediated by Sda beta1,4 N-acetylgalactosaminyltransferase (b4GalNAc-T). The human b4GalNAc-T encodes two transcripts diverging in exon 1: one (long form) encodes a polypeptide with an extremely long cytoplasmic domain of 67 aminoacids while the second (short form) predicts a protein with a cytoplasmic domain of 7 aminoacids. The activity of b4GalNAc-T decreases in colorectal cancer at a very variable extent among patients. Sialyl Lewis x (sLex) is a major ligand of selectins and, if ectopically expressed by tumours, can mediate metastatization. We have permanently expressed the two b4GalNAc-T forms in the human colon cancer cell line LS174T and found that the short form induces a higher level of enzyme activity than the long form. Both forms induce expression of the Sda antigen and inhibit the expression of the sLex, very likely because of the competition between b4GalNAc-T and the fucosyltransferases which synthesize the sLex. The two b4GalNAc-T forms, fused with the green fluorescent protein (GFP) localize largely to the Golgi apparatus although a part of the enzyme molecules was present also in non-Golgi compartments. In normal human colon the Sda antigen is mainly expressed by goblet cells and by the well differentiated enterocytes of the apical portion of the gland. When the level of b4GalNAc-T activity was correlated with sLex expression in colon cancer specimens, we observed a direct relationship instead of an inverse relationship, as expected on the basis of the in vitro study. These data indicate that, although b4GalNAc-T has the potential to inhibit sialyl Lewis x expression, it does not play this role in colon carcinogenesis.

Beta-galactoside alpha2,6 sialyltransferase (ST6Gal.I), the enzyme which adds sialic acid in alpha2,6-linkage on lactosaminic termini of glycoproteins, is frequently overexpressed in cancer, but its relationship with malignancy remains unclear. In this study, we have investigated the phenotypic changes induced by the expression of alpha2,6-sialylated lactosaminic chains in the human colon cancer cell line SW948 which was originally devoid of ST6Gal.I. Clones derived from transfection with the ST6Gal.I cDNA were compared with untransfected cells and mock transfectants. The ST6Gal.I-expressing clones show (1) increased adherence to fibronectin and collagen IV but not to hyaluronic acid. Treatment with Clostridium perfrigens neuraminidase reduces the binding to fibronectin and collagen IV of ST6Gal.I-expressing cells but not that of ST6Gal.I-negative cells; (2) accumulation and more focal distribution of beta1 integrins on the cell surface; (3) different distribution of actin fibers; (4) flatter morphology and reduced tendency to multilayer growth; (5) improved ability to heal a scratch wound; (6) reduced ability to grow at the subcutaneous site of injection in nude mice. Our data suggest that the presence of alpha2,6-linked sialic acid on membrane glycoconjugates increases the binding to extracellular matrix components, resulting in a membrane stabilization of beta1 integrins, further strengthening the binding. This mechanism can provide a basis for the flatter morphology and the reduced tendency to multilayer growth, resulting in a more ordered tissue organization. These data indicate that in the cell line SW948, the effect of ST6Gal.I expression is consistent with the attenuation of the neoplastic phenotype.