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[CANCER RESEARCH 56, 2237-2244, May 15. 1996] Perspectives in Cancer Research Possible Roles of Tumor-associated Carbohydrate Antigens1 Minoru Fukuda2 The Glycobiology Program, La Julia Cancer Research Center. The Burnham Institute, La Jolla, California 92037 the analysis into the roles of carbohydrate-dependent Abstract Recent progress in the studies on the roles of carbohydrates has brought about critical discoveries, which allow us to have working hy potheses for understanding the roles of tumor-associated carbohydrate metastasis. Rather than collect a vast amount of knowledge on changes of cell surface carbohydrates in tumor cells, I have selected these three topics. By doing this, I hope that I will present clear examples that can be a model for working on other systems. Readers are encouraged to read other review articles to see other carbohydrate changes reported in tumor cells (6-9). antigens. In this report, I focus my description on three different aspects of this progress. I discuss: («) the immunologie ¡ilresponse to oligosaccharides aberrantly expressed under pathological conditions; (In possible roles of carbohydrate-dependent cell adhesion during tumor metastasis; and (c) the roles of carbohydrates in modulating the functions of proteins that attach those carbohydrates. These three areas in the roles of carbo hydrates will likely be the target for continuous research efforts to reveal the roles of tumor-associated carbohydrates. Expression of Branched Hexasaccharides Attached to Leukosialin (CD43) during Development and Malignancy Introduction Analysis of cell surface glycoproteins of human leukocytes and leukemic cell lines revealed that the structures of O-glycans aie cell type-specific. Erythrocytes express a tetrasaccharide, NeuNAc «2— »3 Galßl^>3(NeuNAc a.2— >6) GalNAc, whereas granulocytes exptess a hexasaccharide, NeuNAc «2^3 Galßl^»3 (NeuNAc a2^>3 Galßl^4 GlcNAc ßl^6) GalNAc (10). The large mr.jorily of Tlymphocytes in peripheral blood express the same tetn.sacc'-iaride as Cell surface glycoproteins play essential roles in maintaining the function as well as the structure of cells. The importance of cell surface carbohydrates is particularly evident during the course of development and differentiation, as shown by sequential expression and disappearance of cell surface carbohydrates specific to each cell lineage or maturation stage in a given cell lineage (1-4). At the end, mature cells acquire a vast array of different functions and cell surface structures unique to each cell type. In the past decade, a large amount of knowledge has been accu mulated on the carbohydrate structures present in glycoproteins and glycolipids. These studies revealed that those carbohydrates can be classified into several groups, for instance those attached to proteins through aspartylglycosylamine linkage, Af-glycans, and those through O-glycosidic linkage, O-glycans. There appears to be a tremendous erythrocytes express. T-lymphocytes are activated when enga /lag in immune response. Those activated T-lymphocytes now acr/j/e the hexasaccharide (11). This conversion is caused by an incrw.. •¿ i core 2 GnT, which forms a critical branch. In resting T-lymphocy .¿s,the same enzyme is negligibly present, thus forming instead tre tetnsaccharide (Fig. 1). The expression of core 2 GnT suppresses fhf. expres sion of the tetrasaccharide, because this enzyme competes "Atti a-2,6sialyltransferase at the same C-6 position of /V-acetylgalactosamine. In T-lymphocytes, these O-glycans are attached to 1 ul'oJalin heterogeneity among oligosaccharides belonging to the same group, but yet those of apparent complexity can be deduced into the common core structure and the variation in the side chains attached to the core structure (5). One of the most important findings in the past decade is that these variations of oligosaccharides are well controlled during development and differentiation. Thus, oligosaccharides are very often character istic of different cell types. Under pathological conditions such as tumor cells, changes in carbohydrate structures can be almost always observed (6). In this report, I would like to present three examples that exemplify the roles of those oligosaccharides present under pathological condi tions. I will thus first describe the changes in O-linked oligosaccharide structures associated with immunodeficiencies such as WiskottAldrich syndrome and AIDS. In this section, I will discuss immunological response to the altered oligosaccharides and its role in patho logical processes. In the second part, I will concentrate my description on roles of cell surface carbohydrates as ligands and their possible roles in tumor metastasis. In particular, I will summarize the roles of carbohydrates in cell adhesion during inflammation and then extend Received 1/22/96; accepted 3/15/96. 1 Supported by Grants R37CA33000, RO1CA33895, RO1CA48737. adhesion during metastasis. In the third part, I will discuss the roles of carbohydrates as a modulator of protein function. In particular, I will focus ;;n the roles of polysialic acid in the N-CAM3 during development and i mor (CD43, sialophorin). It has been shown that leukosialin is involved in cell-cell interaction: (a) the adhesion of T-lymphocytes to HeLa cells through LFA-1 binding to ICAM-1 was inhibited by expression of CD43 in HeLa cells. This inhibitory activity was abolished, however, when HeLa cells expressing leukosialin was treated with sialidase (12); (¿?) similarly, leukosialin was found to be concenVT;d in the cleavage furrow during cell division (13). Such hig'ìlyi ,;>trictive distribution of leukosialin likely provides repulsion jy v.rative charges due to sialic acid and thus facilitates the dis;, a -.' r. of fwo dividing cells; (c) leukosialin is shed from neutropiii* •¿â€¢ / the spreading and adhesion of activated neutrophils (¡4. -- .tioi!, activated T lymphocytes also shed leukosialin (15). S'K 'I ; of leukosialin appears to be required for cell adhesion, si '..c ; alai leukosialin works as an anti-adhesive molecule. In faci, : . fr tm :i;oof leukosialin is present in the plasma, and its carbo -v sition is consistent in that the leukosialin was derive i cytes and lymphocytes (16). More recent studies suggest that mucin-type O-gly ;. leukosialin play a critical role in thymocyte developrie cells of T lymphocytes, which originated from the boi.'? and POI AI33189 and a grant from the Mizutani Foundation for Glycoscience. 2 To whom requests for reprints should be addressed, at The Glycobiology Program, La Jolla Cancer Research Center, The Burnham Institute, 10901 North Torrey Pines Road. La Jolla, CA 92037. Phone: (619) 455-6480; Fax: (619) 646-3193. 3 The abbreviations used are: N-CAM. neural cell adhesion i-i. W-acetylneuramic acid; GalNAc, W-acetylgalactosamine; GlcNAc. A a core 2 GnT. core 2 ß-l,6-A'-acetylglucosaminyltransferase; ICAM, i molecule; Le, Lewis; PSGL. P-selectin glycoprotein ligand-1; PST, | 2237 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer Research. ROLES OF TUMOR-ASSOCIATED CARBOHYDRATE ANTIGENS GalNAcal -»Servir B1->3Gallransterase Galßl->3GalNAcoc1->Ser/Thr (core 1) ß1 ->6GlcNAc transferase (Core 2 GnT) oc2—>3 NeuNAc Iransferase Galßl->3GalNAca1~>Ser/Trir (core 2) NeuNAco2-»3Galß1 ->3GalNAcce1-»Ser/Thr o2—>6 NeuNAc transferase ß1-»4Gallransferase NeuNAco2 ^6 NeuNAco2-»3Galß1 ->3GalNAca1^->Ser/Thr Fig. 1. The biosynthetic pathways of O-glycans (Ai and polyyV-acetyllactosaminyl O-glycans (B). A. it has been shown that the tetrasaccharide (bottom left) is formed by sequential action of aï—»3 sialyltransferase, followed by ct2—»6 sialyltransferase. When ßl—»6 W-acetylglucosaminyltransferase, core 2 GnT. is present, the branched hexasaccharide (bottom right) is formed (based on Refs. 10, 11, and 22). B, poly-W-acetyllactosaminyl chain can be extended from the GlcNAcßl—*6linkage formed in core 2. Sialylated poly-/V-acetyllactosaminyl extension or W-acelyllactosamine can be further modified by al—»3fucosyltransferase. forming sialyl Lex termini. The core 2 branch is emphasized by a box (based on Fukuda ei al. ( 10), Piller et al.(\\ and Maemura and Fukuda (47)]. a2-»3NeuNActransferasefsi NeuNAco2->3Galß1-»4 NeuNAcot2-»3Galß1 ->3GalNAcoc1-»SerAThr B Galßl->3GalNAca1->Ser;Thr ), jj 1 B1-)3GlcNAc transferase B1->4Gal transferase (Galßl->4GlcN Acß1 ->3)nGalß1 ->4 NeuNAco2->3(Galß1-H.4GlcNAcß1 ->3) Galßl->4 NeuNAca2->3Galß1->3GalN Acal -»Berühr NeuNAco2->3(Galß1->4GlcN Acß1 ->3) „¿Galßl -»4 3 T NeuNAco2-»3Galß1 ->3GalNAca1 -»Ser/Thr Fuc into the thymus for further differentiation and maturation. T-lymphocyte progenitor cells first enter into the periphery of the thymus cortex. The cortical thymocytes (T-lymphocyte precursor cells) thus represent more immature T lymphocytes; then the cortical thymocytes enter into the medulla of the thymus, and they represent more matured T lymphocytes, medullary thymocytes. It was discovered that the cortical thymocytes are strongly stained by a monoclonal antibody, T305, which preferentially reacts with the branched hexasaccharides, NeuNAc a2—>3 Galßl—¿>3 (NeuNAc a2-»3 Galßl->4 GlcNAcßl-»6) GalNAc attached to leukosialin undesirable thymocytes apparently takes place in the medullary thy mus. These results thus suggest a very provocative possibility that the selection for further maturation may be dependent on the turning off of core 2 GnT. Ectopie expression of core 2 GnT in the medullary thymus and its knock-out in the thymus should yield critical informa tion to test this hypothesis. It was also shown that adhesion of thymocytes to thymus epithe lium can be inhibited by T305 antibody ( 17). It has been demonstrated that interaction between thymocyte-thymus epithelium plays an es sential role in thymocyte maturation (19). These results combined together indicate that the interaction between the hexasaccharides on thymocytes and thymus epithelia plays a critical role(s) during thy mocyte maturation in the cortical thymus. Once thymocytes are rel atively matured, they no longer require the hexasaccharides for at tachment to thymus epithelia. It is most likely that the interaction between cortical thymocytes and thymus epithelia is mediated through another interaction, and such an interaction is likely hindered by the presence of the hexasaccharides in leukosialin. (17). In contrast, medullary thymocytes are negligibly stained by the same antibody. Consistent with this finding, the mRNA of core 2 GnT, which forms the core 2 branches, is abundantly present in cortical thymocytes whereas it is not detectable in medullary thymo cytes (17). These results clearly indicate that the O-glycans attached to leukosialin undergo a profound change from the hexasaccharides and larger ones to the tetrasaccharides once thymocytes mature from the cortical thymus to the medullary thymus. It is known that the majority of active thymocytes die before they enter into the medullary thymus. This process is thought to be a Production of Autoantibodies against the Branched typical example of apoptosis. The most recent studies showed that the Oligosaccharides under Pathological Conditions selection in cortical thymus is mainly due to the selection for further In the previous section, I have described the presence of the maturation, and those selected for not being processed for further maturation die in the cortical thymus (18). In contrast, the deletion of hexasaccharides in immature cortical thymocytes and its absence in 2238 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer Research. ROLES OF TUMOR-ASSOCIATED relatively matured medullary thymocytes as well as resting T lym phocytes in the peripheral blood. Once activated, T lymphocytes acquire again the hexasaccharides. This is a typical example in which proliferating matured cells show a phenotype similar to their imma ture precursor cells. This similarity can be extended further to the property displayed by T lymphocytes under pathological conditions: (a) It was shown that leukemic cells derived from T lymphocytes in the peripheral blood are characterized by having a significant amount of the branched hexasaccharides (20, 21). This increase in the hexas accharides is more prominent in those patients with acute lymphocytic leukemia than those with chronic lymphocytic leukemia (20). (b) The increase of the hexasaccharides in the peripheral blood is observed in patients with the Wiskott-Aldrich syndrome (22, 23). The Wiskott-Aldrich syndrome is inherited as an X-linked recessive trait, most likely due to a defect(s) in a single locus of the X chromosome (24). The most recent study identified a gene, which is defective in the syndrome (25). The putative protein product of this gene has the sequence enriched with prolines, which is characteristic for SH3 binding, and a nuclear localization signal. In fact, a mouse homologue of the Wiskott-Aldrich gene, named N-3AP1, was cloned by virtue of its binding to the SH3 domains of the Nek oncogene.4 Nck protein is phosphorylated in response to activation of various cytoplasmic and receptor tyrosine kinases, and Nck has been shown to bind, via its SH2 domain, to activated platelet growth factor and epidermal growth factor receptors (for an example, see Ref. 26). These results indicate that the signal pathways by Nck may be regulated by its binding to the Wiskott-Aldrich gene product. It will be exciting to determine the roles of the Wiskott-Aldrich gene product in the signal pathways. This syndrome is characterized by severe eczema, thrombocytopenia, and susceptibility to opportunistic infection. Patients with this syndrome fail to respond to polysaccharide antigens, rendering them susceptible to bacterial infection. The second critical problem is that T-cell number and function decline quickly with age. T-cell-dependent areas of lymph nodes and spleen becomes progressively depleted (24). This raised the possibility that one of the defects in this syn drome may be the defect in the thymus. Before the advent of better therapy such as bone marrow transplantation, the median life span of these patients was found to be 5.7 years. The cause of death is infection (57% of the total deaths), hemorrhage (27%), and malignant tumors (5%), which are mostly leukemia and Hodgkin's disease (24). From the early stages of the studies, it was discovered that leukosialin behaves abnormally in leukocytes of patients having the Wiskott-Aldrich syndrome. Initially, the studies appeared to suggest that leukosialin is missing in leukocytes from the patients with this syndrome (27). However, it turned out that leukosialin is present but with a high molecular weight form that is the same as that found in activated T lymphocytes (22, 23). It was demonstrated that a substantially increased amount of the branched hexasaccharides are present in leukosialin derived from patients with the Wiskott-Aldrich syndrome. As described above, the hexasaccharides are present in the immature cortical thymocytes but absent in the mature resting T lymphocytes. The expression of the hexasaccharides thus appear to reflect that lymphocytes from the patients with the Wiskott-Aldrich syndrome are functionally imma ture as well. These results strongly suggest that the conversion of the hexasaccharide to the tetrasaccharide is critical for the maturation of T lymphocytes and its failure might induce premature release of immature T lymphocytes from the thymus. Continuous expression of the hexasaccharide is due to the increased amount of core 2 GnT. It is thus tempting to speculate that the 4 W. Li, personal communication (see also Refs. 91 and 92). CARBOHYDRATE ANTIGENS transcription of core 2 GnT is suppressed by the product of the wild-type gene, which is mutated in the Wiskott-Aldrich syndrome. Because of the mutation in the Wiskott-Aldrich syndrome, such gene product may not suppress the core 2 GnT gene expression any longer. During the analysis of lymphocytes from various patients, it was discovered that the peripheral lymphocytes from AIDS patients also contained an increased amount of the hexasaccharide (20). When two different specimens were analyzed, the patient with more full-blown AIDS symptoms showed an increased amount of the hexasaccharide. The patients who were HIV-infected but asymptomatic in AIDS did not show an increase of the hexasaccharide. Interestingly, the sera from HIV-infected individuals were found to contain antibodies reactive with leukosialin, and the sera reacted with leukosialin in thymocytes but not with peripheral blood lymphocytes (28). When peripheral T lymphocytes are partially digested by neuraminidase, the reactivity with the AIDS patients' sera was increased. Leukosialin in thymocytes carry not only the hexasaccharides but also the pentasaccharides, Galßl—»3 (NeuNAc al—»3Galßl—»4 GlcNAcj31^6) GalNAc or NeuNAc a2^3 Gal (Galßl^4 GlcNAcßl—>6)GalNAc, which are partially sialylated oligosaccharides (20). In fact, the amount of Galßl-*3 GalNAc a-2,3-sialyltransferase transcript is more abundant in medullary thymocytes than cortex thymocytes (29). Taken together, these results strongly suggest that AIDS patients produce antibodies against leukosialin, responding to the increased amount of the partially sialylated oligosaccharides present on leuko sialin under such pathological conditions. It has been shown that antibodies to leukosialin can induce bio chemical and functional changes in T lymphocytes in vitro (for an example, see Ref. 30). It is thus tempting to speculate that human anti-leukosialin antibodies in vivo may have similar effects on thy mocytes, resulting in inappropriate activation of thymocytes during the process of maturation. It is presumed that the thymus is required for normal replenishment of CD4+ lymphocytes. Thymic dysfunction thus would be expected to prevent the replenishment of mature CD4+ cells killed by HIV-1. Complementary to the above discoveries, it was found that the HIV-1-infected leukemic T-cell line CEM produced leukosialin that was undersialylated. Such hyposialylation was found to be associated with an impairment of leukosialin-mediated homotypic aggregation (31). Although there has been no report on autoantibodies in the WiskottAldrich syndrome, patients with the Wiskott-Aldrich syndrome may produce similar autoantibodies. If that is the case, those antibodies will likely induce the depletion of thymocytes, which is one of the major symptoms in this syndrome. The presence of autoantibodies in both syndromes could heighten the possibility that these autoantibod ies are involved in the pathogenesis of these diseases. Roles of CD43 (Leukosialin) Revealed by Its Overexpression Gene Knock-out in Mice or The above described studies mostly address the roles of glycans attached to leukosialin. To directly determine the roles of leukosialin expression, overexpression of leukosialin in B-cell lineage and knock out of the leukosialin gene were carried out in mice. As described above, leukosialin is present in early B-cell progeni tors but absent during maturation. Once B lymphocytes differentiate into plasma cells, leukosialin is expressed again. By fusing with the immunoglobulin heavy chain enhancer, leukosialin was continuously expressed in B lymphocytes (32). Those B lymphocytes obtained in the transgenic mice survive longer in vitro and show less sensitivity to apoptosis. These results suggest that leukosialin may deliver a signal 2239 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer Research. ROLES OF TUMOR-ASSOCIATED CARBOHYDRATE ANTIGENS ^¿g^lylylyl,,,^^,^!,^!.^^!^ Fig. 2. Schematic representation of neutrophil and monocyte adhesion to endothelial cells during inflammation. Adhesion of leukocytes is initiated by binding of their carbohydrates (SLe") to E-selectin and P-selectin. Such binding ("rolling effect") leads into firm attachment to endothelial cells by integrin/ICAM interaction and then establishing vascular extravasation through plateletyendothelial cell adhesion molecule 1 (PECAM-1) homotypic interaction. This whole process can be inhibited by administration of sialyl Le*-containing glycoproteins. Tumor cells, in particular carcinoma cells, most likely use this mechanism to adhere to endothelial cells at metastatic sites [from Fukuda (5); see also Springer (36)]. that reduces the number of B lymphocytes that would have normally undergone programmed cell death. When the leukosialin gene was knocked out in mice, the knockout mice developed normal hematopoiesis. However, T lymphocytes from leukosialin-deficient mice showed an accelerated kinetics of T-cell activation than T lymphocytes from wild-type mice (33). This in creased T-cell activation was found to be due to an increase in the proliferation ability of the leukosialin-deficient cells rather than in the number of lymphocytes in the allo-specific precursor pool. To deter mine how these proliferative abilities were achieved, the adhesion of thymocytes and splenic T lymphocytes were investigated. Thymocytes from leukosialin-deficient mice showed substantially increased homotypic adhesion compared with thymocytes from wild-type litter mates. Binding of leukosialin-deficient splenic T lymphocytes was nearly 100% greater than binding of leukosialin-containing cells to fibronectin and 50% greater to ICAM-1. When mice were infected with vaccinia virus, leukosialin-deficient mice showed considerably greater CTL activity. Moreover, infectious virus was recovered from lungs and gonads, which was not observed from wild-type mice. These results suggest that defects involving tissue localization of effector cells or post-lysis viral clearance by phagocytes might occur in the absence of leukosialin. These results, taken together, strongly suggest that leukosialin acts as an antiadhesive property that retards T-lymphocyte interactions. Its carbohydrate moiety, composed of a significant amount of sialic acid, clearly contributes to this function. In this context, the presence of partially sialylated oligosaccharides in leukosialin from the immunodeficient patients may have more significance than they act as immunogenes to produce autoantibodies. Moreover, it is possible that the complexity of those oligosaccharides influences the antiadhesive property of leukosialin and possibly its susceptibility to proteases. In summary, I have described the roles of mucin-type O-glycans attached to leukosialin and leukosialin itself in immunodeficient syn dromes. This field may be one of the few fields where studies have been extensively carried out on both a protein and the glycosylation in a particular glycoprotein. Because of recent isolation of genes that might determine the expression of core 2 GnT, further studies will be expected to provide more insightful and exciting results that would reveal the roles of this interesting molecule. Such studies are expected to shed light on possible roles of autoantibodies formed against tumor-associated carbohydrate antigens. Oligosaccharides in Inflammation and Tumor Metastasis In the past several years, significant progress was made in under standing the roles of oligosaccharides in inflammation. This series of developments was initiated by structural studies on the cell surface carbohydrates of neutrophils. These early studies demonstrated that neutrophils contain Le\ GaljSl —¿*4(Fucal^>3)GlcNAc—>R and sia lyl Le", NeuNAca2-*3 Galßl^4(Fucal^»3)GlcNAc^R (34, 35), but not blood group ABO antigens, which are the major oligosaccha rides present in erythrocytes. In 1989, the cDNAs for three different adhesion molecules, now collectively called "selectin," that bind to neutrophils and monocytes or endothelial cells were cloned. Among them, E- and P-selectin appear in the onset of inflammation, and binding of these selectins to neutrophils and monocytes results in the recruitment of these leuko cytes into inflammatory sites (reviewed in Ref. 36). Since all of these selectins were found to contain a carbohydratebinding motif observed in animal lectins, it was natural to test if selectins bind to carbohydrates. These studies revealed that E- and P-selectin binds to sialyl Le", present as a minor component in neutrophils (37-39). This binding of E-and P-selectins slows down the movement of neutrophils along the endothelial wall, which then allows leukocytes to have enough access to chemokines. Once chemokines are bound to their receptors on leukocytes, a signal is presumably transmitted through G-proteins, which activates integrins on the surfaces of leukocytes, establishing a firm attachment to endothelia. This firm attachment then leads into extravasation, possi bly through homophilic interaction of platelet/endothelial cell adhe sion molecule 1 (Fig. 2). It is possible that sialyl Le" and E-selectin interaction also plays a role in cell-cell interaction during develop ment. In fact, it has been shown that the interaction of E-selectin and sialyl Le" is involved in capillary tube formation from bovine endo thelial cells (40). Tumor cells, in particular carcinoma and leukemic cells, are enriched with sialyl Le" structures. Carcinoma cells are also enriched with sialyl Lea structure NeuNAcc*2^3Galßl^3(Fucal->4)GlcNAc^R, which is the isomer of sialyl Lex (41, 42). Sialyl Lea was found to be a ligand for E-selectin, which is as potent as sialyl Le" (43, 44). Considering a similar carbohydrate binding specificity between E- and P-selectin, Pselectin might bind to sialyl Lea as well. Although E- and P-selectin bind to these oligosaccharides, 2240 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer Research. these ROLES OF TUMOR-ASSOCIATED two selectins differ in the requirement for how these oligosaccharide ligands are presented. It has been observed repeatedly that E-selectin binds to cells as long as they express sialyl Le" structures. However, P-selectin binds to sialyl Lex structures only when they are attached to particular glycoproteins. One of these carrier glycoproteins, PSGL-1, was cloned by the virtue of its support of high affinity binding to P-selectin (45). The amino acid sequence of PSGL-1 was found to contain a domain composed of tandem repeats that should attach a large number of mucin-type O-glycans. Similarly, the high affinity ligand for L-selectin is presented by two mucin-type glycoproteins, CD34 and GlyCAM-1 (46). These results strongly suggest that P- and L-selectins bind mucin-type O-glycans that are present as a cluster. It is likely that the multiple presentation of ligands leads into high affinity binding of P- and L-selectin. In contrast, E-selectin apparently binds to the carbohydrate ligands, regardless of how they are pre sented (37). For the synthesis of the sialyl Le" structure in mucin-type oligosaccharides, the presence of core 2 GnT is essential (Fig. 1; Ref. 47). Only those cells expressing core 2 GnT can present efficient ligands for P- and L-selectin. In some instances, it may even be important to have sialyl Le" in mucin-type oligosaccharides for E-selectin. For example, activated T-lymphocytes adhere to E-selectin whereas rest ing T lymphocytes do not (48). As described above, activated T lymphocytes express core 2 GnT, thus allowing the expression of sialyl Le" in mucin-type oligosaccharides present in activated T lymphocytes. These results strongly suggest that the expression of the branched hexasaccharide and its undersialylated forms widely play roles in cell-cell interaction. Since both tumor cells and neutrophils contain sialyl Lex on the cell surface, it is an attractive hypothesis that tumor cells use the same selectin-carbohydrate interaction during metastasis. When tumor cells disseminate, blood-borne tumor cells must adhere to endothelial cells at metastatic sites (49). Once tumor cells adhere to endothelial cells, they then penetrate through endothelial cells, moving into subendothelial tissues. Once tumor cells grow beneath the endothelia, tumor metastasis is established (see also Fig. 2). In fact, it was shown in early studies that tumor cells bind to endothelial cells by E-selectin/ carbohydrate interaction (50). It has been demonstrated that highly metastatic colonie carcinoma cells bind more strongly to E-selectin expressed on activated, human endothelial cells than low-metastatic counterparts (51). A similar difference was found in the binding of the same colon carcinoma cells to mouse endothelial cells that were activated (51). The latter findings indicate that metastasis in nude mice mostly reflects tumor metastasis in humans, at least at the level of E-selectin-mediated adhesion. Poorly metastatic tumor cells expressing low levels of sialyl Le" structure were genetically engineered to increase the amount of sialyl Le". Resultant cells were found to adhere more strongly to E-selectins (52). More importantly, it was discovered that the amount of sialyl Le" on colonie carcinoma cells is directly correlated to the prognosis CARBOHYDRATE ANTIGENS contains not only sialyl Le" but also sulfated tyrosine, which appears to play critical roles for the binding of PSGL-1 to P-selectin (54-56). Consistent with this result, it was reported that different kinds of tumor cells bind more efficiently to P-selectin after the cells were transfected to express PSGL-1 (57). These tumor cells apparently contain sulfated glycans that inhibit sialyl Le" and sialyl Le"-dependent adhesion to P-selectin (58). However, a recent report indicates that colonie carcinoma cells, the same cells used in the above studies (51, 52), can adhere to P-selectin under flow conditions (59), which should be closer to the actual situation when tumor cells adhere to platelets. These colonie tumor cells were shown to lack PSGL-1 (59). It is thus possible that tumor cells express a P-selectin ligand presenter(s) that is different from PSGL-1. (b) In tumor cell binding to selectins, which /V-glycans or mucintype glycans primarily carry high-affinity ligands for selectins? In this regard, it is interesting to note that colo 205 colonie carcinoma cells express sialyl Le" in mucin-type glycoprotein leukosialin, which may be involved in E- and P-selectin-mediated adhesion (60). (c) How can the amount of sialyl Le" in tumor cells be correlated to the presence of other carbohydrates present in tumor cells'? For example, the presence of sialyl Tn, NeuNAca2—»6GalNAcal—»Ser/ Thr or Tn antigen GalNAcal—»Ser/Thr in mucin-type oligosaccha rides likely indicates that the mucin-type O-glycans present in those tumor cells are shorter than those that do not express sialyl Tn or Tn antigen (61). If this is the case, those tumor cells likely contain less amounts of sialyl Le" and sialyl Le" structures. It is then possible that colonie carcinoma cells expressing sialyl Tn or Tn antigen is less metastatic than those that express less of the antigen. Contrary to this, it was reported that compared to patients with colonie tumors con taining less amounts of sialyl Tn and Tn, those containing increased amounts of these antigens showed a poorer prognosis (62). Further studies are thus necessary to clarify this point. Those studies address ing the above three points should provide a comprehensive under standing on the roles of tumor-associated carbohydrate antigens dur ing metastasis. It is also necessary to mention here that carbohydrate-protein in teraction may play a role beyond the scope of selectin-carbohydrate interaction. In fact, it has been shown that galectin-1 on endothelial cells, which belongs to a family of S-type lectin, is involved in apoptosis during T-cell development (63). It is noteworthy that the binding of galectin-1 to /V-glycans on thymocytes is critical in this process. Mucin-type O-glycans are inhibiting this process (63). Con sidering that the conversion of O-glycans from the hexasaccharide to the tetrasaccharide in thymocyte development (17), it is likely that bulkier O-glycans are more efficient in inhibiting the interaction between galectin-1 and iV-glycans. These results suggest that carbo hydrate-protein interaction in tumor metastasis may also use mecha nisms other than selectin-carbohydrate interaction. Further studies will be of great significance to reveal such interactions. of the patients with these carcinomas (53). The patients with a higher expression of cell surface sialyl Le" structures display much poorer prognosis than those with lower expression of sialyl Lex, although Modulation of the N-CAM by Polysialic Acid other histological criteria were apparently similar. These combined studies strongly suggest that one of the key factors in metastatic spread is the abundance of sialyl Le" structure and possibly sialyl Lea, the ligand for E- and P-selectin expressed in tumor Polysialic acid is a developmentally regulated carbohydrate com posed of a linear homopolymer of a-2,8-linked sialic acid residues. Polysialic acid is mainly attached to the N-CAM. Although polysialylated N-CAM is abundant in embryonic tissues, the majority of N-CAM in adult tissues lacks this unique glycan (64-66). The pres cells. There are several points that need to be understood: (a) It has not been determined whether tumor cells bind to Pselectin as well. This is a critical point since tumor cells aggregate around platelets (49) and there is a possibility that this aggregation is due to the interaction of P-selectin on platelets and carbohydrates on tumor cells. In this regard, it is important to point out that PSGL-1 ence of this large negatively charged carbohydrate modulates the adhesive property of N-CAM, and removal of polysialic acid in creases binding between N-CAM-bearing liposomes and neuroblas toma cells expressing N-CAM (67, 68). During embryonic develop ment, the polysialylated, embryonic N-CAM is restricted to specific cell types where cells are migrating (69, 70), and the removal of 2241 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer Research. ROLES OF TUMOR-ASSOCIATED polysialic acid from the N-CAM was reported to influence motor neuron projections in the embryonic tissues (69). Other studies sug gest that the polysialylated form of N-CAM not only exhibits antiadhesive properties on homophilic N-CAM interaction but also attenu ates cell-cell interactions carried by other cell adhesion molecules (65, 71, 72). The recent studies of N-CAM knock-out mice demonstrated a defect in spatial learning and memory, due to an anomaly of the olfactory bulb and hippocampus (73 74), where polysialic acid is continuously expressed also in adult brain (75, 76). These results strongly suggest that polysialylation regulates the function of NCAM. Due to the presence of polysialic acid in these tissues under going synaptic rearrangement and cell migration, polysialic acid is implicated in reducing N-CAM adhesion and thus perhaps allowing increased neurite outgrowth and cellular mobility. Although the expression of polysialic acid is diminished in the majority of tissues in the adult, some tumors are known to re-express polysialic acid; thus, polysialic acid represents an oncodevelopmental antigen. The first example of this expression was discovered in Wilms' tumor (77). Wilms' tumor is a primary renal tumor of child hood and is characterized by the presence of structural elements, which are found during embryonic development of the human kidney. In particular, undifferentiated blastoma regions, which are composed of small closely packed cells, express the highest amount of polysialic acid. Regions showing epithelial differentiation, such as glomeruloid structures and tubules, exhibited only a weak staining. The majority of these polysialic acid glycans were found to be attached to N-CAM. These results clearly establish that Wilms' tumors express polysialic acid, which is suppressed in normal adult kidney. Very recently, a cDNA encoding a PST was cloned (78, 79). The predicted amino acid sequences of both human and hamster PST show that the enzyme consists of 359 amino acid residues and has homology with other sialyltransferases belonging to the sialyltransferase superfamily. It was also demonstrated that PST can form all a-2,8sialic acid residues necessary for polysialic acid formation (80). Consistent with the above results on tissue distribution, mRNA of PST is substantially present in fetal kidney but absent in adult kidney (78). It will be thus of interest to test if the amount of the PST transcript is elevated in Wilms' tumor. It will be also significant if Wr-1, the tumor suppressor gene identified in Wilms' tumor (81), regulates the gene expression of PST. Small cell lung carcinoma is a highly malignant tumor showing some neuroendocrine characteristics. It was found that human small cell lung carcinomas, irrespective of their histológica! type, express polysialic acid (82). Metastatic tumor cells, derived from small cell lung carcinomas, also express polysialic acid. In contrast, polysialic acid was not detected in the bronchial as well as gastrointestinal carcinoids. In one of the cell lines derived from a human small cell lung carcinoma (NCI-H69), a subline containing a negligible amount of polysialic acid (E2) and that containing a significant amount of polysialic acid (F3), respectively, were established (83). A most recent study showed that the expression of polysialic acid in this small cell lung carcinoma cell line is directed by sialyltransferase X, which is highly homologous to PST (84). In the majority of other cells, it appears that PST determines the expression of polysialic acid (78). However, these results indicate that it is necessary to determine whether PST or sialyltransferase X directs the expression of polysialic acid in a given cell (see Refs. 80 and 84 for detailed discussion). When these two carcinoma sublines, derived from NCI-H69 as described above, are compared, F3 shows a much more retarded reaggregation than E2, after cells were dissociated and let them aggregate. This retarded reaggregation disappears after removal of polysialic acid by endoneuraminidase. More importantly, compared to the polysialic acid-negative clonal CARBOHYDRATE ANTIGENS N-CAM + PSA Fig. 3. Neurite outgrowth dependent on polysialic acid attached to N-CAM. Parent HeLa cells. HeLa cells stably expressing N-CAM alone, and HeLa cells expressing N-CAM and polysialic acid were cultured as monolayers. Neurons from embryonic chicken were seeded on those HeLa cells and cultured for 15 h. The neuntes were visualized by immunofluorescent staining for neurofilament. Neurons cultured on HeLa cells expressing N-CAM and polysialic acid grew significantly longer neuntes, which exhibited more branching. AAA. polysialic acid [based on Nakayama el al. (78)]. subline, (E2), polysialic acid-positive clonal subline (F3) forms much more colonies in soft-agar or methyl cellulose. Most significantly, i.e. metastasis took place when F3 subline was s.c. injected into nude mice. In contrast, the polysialic acid-negative subline produced only a negligible amount of metastasis, despite the fact that both E2 and F3 express comparable amounts of N-CAM (83). These results strongly suggest that masking or weakening of NCAM homophilic or heterophilic interaction may allow those tumor cells to detach more easily from primary tumor. They also may be able to survive in the blood stream by preventing the attachment to endothelial cells. Consistent with these discoveries, the amount of N-CAM itself is reduced in some tumor métastases(85, 86). In these tumors, either decreasing of N-CAM itself or weakening the adhesive capability of N-CAM by polysialic acid results in increased metastasis. When tumor cells migrate and adhere to certain tissues, polysialic acid-containing N-CAMs appear to play roles. Natural killer cellderived lymphomas express polysialylated N-CAM, and their metastatic patterns are characterized by unusual tissue involvement, such as the central nervous system, nasopharyx, gastrointestinal tract, skel etal muscle, and kidney (87, 88). The recent studies showed that mRNA of PST can be detected in all of these tissues (78). Moreover, those tissues express polysialic acid detected by anti-polysialic acid antibody. These results strongly suggest that lymphoma cells express ing polysialic acid and N-CAM migrate to the tissues where N-CAM and polysialic acid are expressed, achieving the binding of those lymphoma cells at the metastatic sites. For successful attachment of lymphoma cells, however, the amount of polysialic acid should be moderate in both lymphomas and tissues where the attachment takes place. If the polysialic acid content is too high, the detachment of lymphoma cells can take place, but the attachment of those cells to particular tissues may not be possible. Although there is no quantita tive data of the polysialic acid content on lymphoma cells, it is noteworthy that the heart, which expresses a very high amount of polysialic acid (78), is not a preferential site for the attachment of lymphoma cells. It has been shown clearly that neurite outgrowth is facilitated much better on substrate cells that express both N-CAM and polysialic acid than those expressing N-CAM alone (Fig. 3; Ref. 78). The presence of polysialic acid thus allows cells to migrate more efficiently, prevent ing static adhesion (89). Such modulated cell movement may be also critical when tumor cells eventually adhere to the sites where they 2242 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1996 American Association for Cancer Research. ROLES OF TUMOR-ASSOCIATED grow, after invading into subendothelial tissues and further migrating into the metastatic sites. Further studies will be important to determine the roles of polysialic acid by testing the metastatic capability of tumor cells that express a different amount of polysialic acid by gene manipulation. In this regard, it is also now possible to determine the roles of polysialylated gangliosides by using a cDNA encoding the newly identified ganglioside-specific polysialyltransferase (90). In summary, I have described three different roles of tumor-asso ciated carbohydrates in the pathogenesis of tumor formation. The first section concerned immunogenicity toward oligosaccharides that are aberrantly synthesized. The second section concerned the ligand func tion of oligosaccharides in protein-carbohydrate interaction. The third CARBOHYDRATE ANTIGENS 19. Patel, D. D.. and Haynes. B. F. Cell adhesion molecules involved in intrathymic T cell development. Semin. 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