Schwab (ed.), Encyclopedia of Cancer, DOI 10.1007/978-3-642-27841-9_2457-2
Carbohydrate part of glycoconjugates
Glycosylation is defined as the covalent attachment of a carbohydrate to a protein, lipid, carbohydrate, or other organic compounds, catalyzed by glycosyltransferases, utilizing specific sugar donor substrates.
The type of glycosylation of the different biomolecules can be used to classify the following families of glycoconjugates: the asparaginelinked (N-linked) oligosaccharides of many glycoproteins; the serine or threonine-linked (O-linked) oligosaccharides that are present on many glycoproteins and that predominate on secreted or membrane-bound mucins; the glycosaminoglycans (GAG), which are glycans present as free polysaccharides or as part of proteoglycans (such as heparin sulfate and chondroitin sulfate); the glycosphingolipids, which consist of oligosaccharides linked to ceramide; the glycosylphosphatidylinositol (GPI)-linked proteins, which are proteins that bear a glycan chain linked to phosphatidylinositol; and nuclear and cytoplasmic proteins, which bear the monosaccharide O-linked N-acetylglucosamine (O-GlcNAc) linked to serine. Glycans can exist as membrane-bound glycoconjugates, for example, forming the glycocalix, or as secreted molecules, for example, as components of the extracellular matrix. Glycans mediate key cellular functions such as cell–cell interactions, adhesion, and motility, as well as intracellular signaling events.
Glycosylation alterations and functions in cancer
Altered glycosylation is a common feature of cancer cells. These modifications have been observed both histologically using lectins or monoclonal antibodies and by mass spectrometry. Changes in glycosylation in cancer include both underand overexpression of naturally occurring glycans, as well as novel expression of glycans normally restricted to other adult tissues, or structures that arise during development in embryonic tissues. The biosynthesis of these altered glycans generally arises from changes in the expression of the glycosyltransferases. Glycosyltransferases are Golgi-resident enzymes that are responsible for the biosynthesis of oligosaccharide chains. Modification of the set and the level of expression of glycosyltransferases can lead to changes in the structures of protein N-linked and O-linked glycans as well as glycolipids. Glycans play main roles in the biology of the tumor cells.
Гликозилирование и клеточный сигналинг
N-Linked glycans participate in the control of protein folding and play a crucial role in the protein half-life and quality control systems. N-Glycans may also interfere cellular receptors and tumor growth. N-Glycosylation on insulin-like growth factor 1 receptor (IGF1R) affects its phosphorylation and therefore influences cell-surface receptor translocation, leading to modification of the signaling and affecting the growth and survival of melanoma and sarcoma cells. Inhibitors of N-glycosylation can reduce the survival of cells from tumors that depend on IGF1R signaling.
Glycosphingolipids, particularly gangliosides frequently overexpressed in tumors, are also involved in cell signaling and tumor cell growth. Gangliosides are capped with sialic acid, a negatively charged acidic saccharide, on the terminal tip of the glycan. In normal cells, gangliosides are often associated with certain cell membrane tyrosine kinases receptors, such as EGF receptor and IGF receptor, modulating their phosphorylation and interfering with their growth-promoting functions. The ganglioside GM3, which is commonly expressed in lung cancers, melanomas, and neurogenic tumors, is able to regulate growth signaling through the receptor tyrosine kinases. Gangliosides also contribute for the molecular organization of receptor complexes in lipid rafts. Ganglioside GM1 contributes for ERBB3 and ERBB4 receptors to form heterodimers on lipid rafts on breast cancer cell membranes, facilitating ERBB signaling and functions.
Another good example of glycoconjugates interfering with cell signaling and promotion of proliferation comes from mucins. Mucins are secreted or membraneassociated glycoproteins that contain clusters of O-glycans in a central domain of the core protein. The mucin (MUC) gene family includes several members of glycoproteins that are expressed in a tissueand cell-specific manner. Most carcinoma expresses mucins with altered O-glycan chains. Membrane-associated mucins can interfere with signal transduction. MUC4 mucin is overexpressed in mammary tumors and contains an epidermal growth factor (EGF)-like motif on its extracellular (juxtamembrane) domain which interacts with the ERBB2, initiating phosphorylation of the receptor tyrosine kinase in the absence of other ligands. In breast carcinomas, ERBB2 is frequently overexpressed or mutated in an active form. Experimental studies have showed that MUC4 overexpression increases cell growth and that MUC4overexpressing cells can autophosphorylate ERBB2, contributing for the inhibition of apoptosis. O-Glycans on MUC4 play an important role in the protein folding leading to the exposure of the EGF-like domain and its activity in cancer cells. Another membrane-associated mucin, MUC1 shows an altered O-glycosylation carrying several cancer-associated carbohydrate antigens. The cytoplasmic tail of MUC1 can be phosphorylated following mucin interactions with other partners. This may modify signaling pathways and affect b-catenin signal transduction by two mechanisms: MUC1 might be cleaved in the cytoplasm and move with b-catenin to the nucleus, thereby influencing transcription, or it might sequester b-catenin in the cytosol and therefore prevent it from interacting with the cadherins or other complexes, thereby influencing WNT signaling. Mucin O-glycans are also involved in the invasive and metastatic properties of adenocarcinomas by simultaneously configuring the adhesive and antiadhesive cell-surface properties of tumor cells. Mucin O-glycans contain domains that mediate both antiadhesion and adhesion effects and might block proteolytic activity.
Proteoglycans may function as coreceptor for soluble growth factors
The structure of proteoglycans consists of a core protein and one or more GAGs. GAGs are linear polysaccharides composed of repeating disaccharide units (GlcNAc in heparan sulfate or GalNAc in chondroitin sulfate) linked to uronic acid (GlcA or IdoA). Most mammalian cells produce proteoglycans that may be secreted to the extracellular matrix, inserted into the plasma membrane, or stored in secretory granules. GAG binding to a protein ligand may cause (i) immobilization of the protein ligand at site of production, (ii) interference with enzyme activity, (iii) interaction with signaling receptors, (iv) protection of ligands against degradation, and (v) reservation of ligand for future mobilization. These interactions lead to profound physiological effects, affecting processes such as cell–matrix (interaction with fibronectin, laminin, and thrombospondin), growth (interaction with fibroblast growth factors, transforming growth factor-b), angiogenesis (interaction with vascular endothelium growth factor), and inflammation (interaction with selectins, interleukin-8, and others). The tumor microenvironment often shows significant alterations in content of proteoglycans. Cancer cells seem to usurp GAG-mediated functions to promote tumor growth, progression, and invasion. For example, breast cancer, ovarian, pancreatic cancer, and hepatocellular cancer cells modulate sulfation of proteoglycans in a manner that promotes their binding capability for growth factors and inducing receptor tyrosine kinase activation.
Glycans in tumor cell adhesion and invasion
Nand O-glycosylation of glycoproteins controls different tumor cell biological properties. One of the best studied modification observed in cancer cells is the increased expression of complex b1,6-branched N-linked glycans on their cell surface. This alteration is caused by an increased expression of the enzyme N-acetylglucosaminyltransferase V (GnTV). This enzyme transfers a GlcNAc residue onto growing N-linked glycans so that the subsequently glycosylation results on multiantennary chains. This increased branching creates additional sites for terminal sialic acid residues, which, together with the corresponding upregulation of sialyltransferases, may lead to an increased sialylation. The expression of complex b1,6-branched N-glycans has been shown on tumor cell E-cadherin – an adhesion molecule that mediates cell adhesion through homotypic interactions and b1-integrin. Increased b1,6branched N-glycans on tumor cell E-cadherin reduces cell–cell adhesion and promotes tumor cell detachment and invasion. The coordinated control of expression of GnTV and other molecules, such as E-cadherin, contributes for the modulation, adhesion, and invasion phenotype of tumor cells.
Alterations in O-glycosylation are common feature observed in glycoproteins expressed in tumors cells. These alterations include the expression of simple mucin-type O-linked carbohydrate antigens, such as the Tn, sialyl-Tn, T, and sialyl-T antigens, which are rarely expressed in normal cells. The biosynthesis of these glycans may occur due to different reasons, such as altered expression and cellular mislocalization of glycosyltransferases, imbalanced competition among glycosyltransferases for acceptor substrates, and overexpression of acceptor substrates. The sialyl-Tn antigen is a good example of a simple O-linked glycan that is abnormally expressed in membrane-associated and secreted glycoproteins, mainly mucins, from tumor cells. Sialyl-Tn expression is associated with an aggressive phenotype. Transfection of gastric cancer and breast cancer cells with the cDNA that encodes the sialyltransferase responsible for
sialyl-Tn biosynthesis (ST6GalNAc-I) modifies cell–cell aggregation and increases migration and the cell invasion potential. These observations are in agreement with the association of sialyl-Tn expression and the increased metastatic potential and poor prognosis observed in colorectal, gastric, and ovarian carcinoma.
In addition to changes in the core structures of glycans, alterations of terminal structures are also associated with malignant phenotype. Glycosyltransferases involved in the biosynthesis of terminal structures, such as sialyltransferases and fucosyltransferases, frequently show altered expression in tumors. The altered expression of these enzymes leads to overexpression and/or neoexpression of terminal glycans, namely, sialyl-Lewis X (S-Lex) and sialyl-Lewis A (S-Lea), among others. These terminal structures can be carried on Oand N-linked glycans on proteins as well as on glycolipids.
The expression of S-Lex and S-Lea in cancer cells can promote metastasis through the interaction with selectins. Selectins normally mediate adhesion between leukocytes (which express L-selectin), platelets (P-selectin), and endothelial cells (Eand P-selectins) that bear the appropriate glycosylated ligand. This ligand consists of oligosaccharides containing the terminal S-Lex structure. The presence of such ligand on blood cells (such as monocytes or neutrophils) or in endothelial cells induces the transmigrations of leukocytes to sites of inflammation and/or infections, as well as of platelets to sites of vascular damage. In contrast to their normal counterpart, tumor cells from gastric cancer, colon cancer, pancreatic cancer, and lung carcinoma frequently overexpress S-Lex or S-Lea on glycoproteins and glycosphingolipids. The expression of these glycans is associated with poor prognosis for the patients. These glycans have been shown in metastatic tumor cells in the blood of colon carcinoma patients and demonstrated to bind to E-selectin. Likewise, overexpression of E-selectin in a transgenic mouse liver induced redirection of the metastatic patterns of syngeneic carcinomas that normally metastasize the lung. Furthermore, the use of a specific disaccharide decoy that acts as a competitive substrate of glycosyltransferases reduced the levels of S-Lex on colon carcinoma cells. In comparison to untreated cells, these S-Lexdeficient cells showed decreased interactions with selectins, increased susceptibility to leukocyte-mediated lysis, and reduction of lung metastasis in a murine tumor model. These studies, together with the clinical data, indicate that interactions between glycans and selectin molecules play a role in the metastatic process of some carcinoma cells. In summary, this may fit with the classical observation that cancer cells entering the bloodstream tend to form thromboemboli with platelets and leukocytes, probably facilitating the transport to distant sites, contributing to evasion of the immune system, and finally participating in the process of extravasation in the distant metastatic site.
Alterations in the levels of sialylation, namely, oligosaccharide chains containing terminal sialic acid, are also often observed in tumor cells. This is the case of sialic acid capping terminal galactose in N-linked glycans by the enzyme ST6GalI, which is upregulated in colon and breast cancers. Hypersialylation of glycans on b1-integrins augment colon tumor progression by altering cell preference for certain extracellular matrix milieus, as well as by stimulating cell migration. In addition, polysialylation controlled by polysialyltransferases can modulate the sialylation of molecules, such as NCAM in human tumor cells, which has been linked to cancer development and dissemination.
Glycans used as serological markers in cancers
The most widely used serum diagnostic assays for carcinomas are based on serum measurement of certain glycans. These serological assays are currently used to facilitate diagnosis, track tumor recurrence or tumor burden, or provide a surrogate measure for therapeutic response. For example, the serological marker CA125 detects the MUC16 glycoprotein, and its detections in the serum of ovarian cancer patients are associated with tumor burden and prognosis. Another useful serological assay is the CA19-9, which recognizes the S-Lea carbohydrate antigen present on mucins and glycolipids. CA-19-9 can be used in pancreatic, gastric, and colorectal carcinomas. The CA15-3 assay, which recognizes the MUC1 mucin glycoprotein, is also a useful marker commonly overexpressed in breast carcinomas with serum levels also associated with tumor burden and prognosis. In an attempt to improve their clinical application of the serological assays, a combination of the existing assays against carbohydrate structures detected in the serum have improved the sensitivity with a concomitant loss of specificity. Other assays with higher specificity are under investigation.
Therapeutic applications of glycans in cancers
Various therapeutic approaches are being developed targeting glycans. These strategies include the targeting of glycans that affect tumor proliferation, such as the interference with the coreceptor activity of proteoglycans on tumor cells. In addition, various approaches are targeting glycan during invasion using methods to block specific Nand O-glycosylation steps as well as the use of competitors of proteoglycans that once again interfere with tumor cell invasion. Tumor cell dissemination is another step in which glycans can be targeted. Agents that can interfere with selectin – carbohydrate interactions have been considered for the treatment of metastatic disease. These interactions can be targeted through the administration of neutralizing antiselectin antibodies or small molecules that mimics the SLeX or SLeA selectin ligands. The applicability of such experimental therapies to clinical tumor biology and metastasis remains challenging, but the importance of this strategy warrants further development of this class of therapeutic agents, which might work best in combination with conventional chemotherapy.
In addition, several immunotherapy-based approaches are being tested in preclinical and clinical trials targeting cancer-associated mucin glycans expressed on adenocarcinomas. These trials include monoclonal-antibody-based therapies – either radioimmunoconjugates or passive immunotherapy and antitumor vaccines. Monoclonal antibodies directed to cancer-associated glycans, such as S-Tn, and glycan carriers, such as MUCl mucin, have been used as radioimmunoconjugates. Second-generation approaches are under development, such as the use of humanized forms of the murine antibodies as well as recombinant single-chain variable fragments, which have a much smaller mass than full antibodies and therefore are capable of reaching most cells in a tumor.
Another immunotherapy-based approach aims at augmenting the immunity against tumor mucin glycans. A tumor’s ability to generate and overexpress unique mucins and glycans is now being used to improve or augment the immune system’s ability to recognize and destroy tumor cells. Methods to destroy tumor cells using vaccines that target the tumor mucins MUC1 or MUC16, or the cancer-associated S-Tn glycan, have been introduced. Vaccines of this type are typically prepared by conjugating the glycan to a carrier protein and then injecting the compound into the patient together with an adjuvant that boosts T-cell responses. Vaccines that contain these epitopes are currently being used in breast, colorectal, and ovarian cancer patients to augment antitumor immune responses. In addition, peptide mimetics of tumor carbohydrates, such as SLeX, SLeA, or SLeY, have also been shown to stimulate tumor immunity in animal models. In another promising strategy, antitumor immunity has been generated by making unique alterations to tumor cell surface sialic acids (which are normally tolerated by the immune system). This might be accomplished by introducing unnatural sialic acid precursors to tumors whereby metabolic incorporation leads to the generation of unnatural sialic acid epitopes, forming the basis for the induction of novel antitumor immune responses.
Other approaches have targeted at augmenting immunity against tumor glycosphingolipids. Some tumors express very high levels of immunogenic gangliosides and often shed them into the bloodstream. Paradoxically, this can lead to immune silencing, by inhibition of costimulatory molecule synthesis and by the arrest of dendritic cell maturation, resulting in the inability to generate effective antitumor T-cell immune responses. Although glycosphingolipids are relatively poor immunogens, certain glycosphingolipids might be manipulated to generate both passive and active immunity by eliciting a host response to a tumor glycan. Immunization with purified GD2 or GM2 gangliosides are being used in current clinical trials against melanoma (using GD2–KLH), neuroblastoma (using anti-GD2 small immunoproteins), breast cancer (using NeuGcGM3 proteoliposomes that contain the N-glycolyl form of sialic acid), and prostate carcinomas (using a conjugate between KLH and Globo-H hexasaccharide, a glycan expressed on prostate and breast cancer glycolipids).
Finally, liposomal drug-delivery techniques such as antisense oligodeoxynucleotides directed to inhibit the synthesis of oncogenes have been delivered to human neuroblastomas by encapsulation in cationic liposomes that have been covalently coupled to monoclonal antibodies against the GD2 ganglioside expressed in the tumor cells.
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