K. Sugahara, Laboratory of Proteoglycan Signaling and Therapeutics, Frontier Research Center for Post-Genomic Science and Technology, Hokkaido University Graduate School of Life Science, West-11, North-21, Kita-ku, Sapporo, Hokkaido 001-0021, Japan
Glycosaminoglycans, including chondroitin sulfate (CS), dermatan sulfate, and heparan sulfate, attached to proteoglycans at the surface of tumor cells play key roles in malignant transformation and metastasis. A Lewis lung carcinoma (LLC)-derived tumor cell line with high metastatic potential shows a higher proportion of E disaccharide units, d-glucuronic acid–GalNAc(4,6-O-disulfate), in CS chains than LLC cells with low metastatic potential, suggesting that E units in the CS chains contribute to the metastatic potential. In fact, the metastasis of LLC to mouse lungs is drastically inhibited by preadministration of CS-E or a phage display antibody specific for CS-E. However, the molecular mechanism underlying the pulmonary metastasis involving CS chains remained to be elucidated. Recently, receptor for advanced glycation end-products (RAGE), which is predominantly expressed in the lung, was identified as a functional receptor for CS chains containing E units. RAGE strongly interacted with not only CS-E but also heparan sulfate in vitro. The interaction with CS-E required a decasaccharide length and a cluster of basic amino acids. Intriguingly, antibody against RAGE robustly inhibited the pulmonary metastasis of not only LLC but also B16 melanoma cells, which also colonize mouse lungs after injection into a tail vein. Thus, CS chains containing E units are involved in the metastatic process, and RAGE is a critical receptor for glycosaminoglycan chains expressed at the tumor cell surface. Hence, RAGE and glycosaminoglycans are potential targets of drugs for pulmonary metastasis and a number of other pathological conditions involving RAGE in the pathogenetic mechanism.
Chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulfate (HS) chains are covalently attached to various proteoglycans (PGs) as side chains. These glycosaminoglycan (GAG) chains are ubiquitously distributed at cell surfaces and in extracellular matrices. PGs function through the GAG side chains in various physiological processes, such as cell behavior, tissue morphogenesis, neurite outgrowth, infection with viruses/bacteria, inflammation, and the regulation of various growth factors and cytokines [1-5]. CS and DS chains are linear polysaccharides composed of disaccharide units, (4GlcUAβ1–3GalNAcβ1)n and (4IdoUAα1–3GalNAcβ1)n, respectively (where GlcUA and IdoUA stand for d-glucuronic acid and l-iduronic acid, respectively), which are modified by sulfation at various hydroxy groups on each sugar residue. They are often distributed as CS/DS hybrid chains in mammalian tissues, and are modified by specific sulfotransferases, which transfer a sulfate group from 3′-phosphoadenosine 5′-phosphosulfate, the universal sulfate donor, to C-2 and/or C-3 of GlcUA and/or C-2 of IdoUA, as well as C-4 and/or C-6 of GalNAc residues to yield enormous structural diversity. These enzymes are regulated in a spatiotemporally specific manner, giving rise to structural diversity resulting from combinations of various characteristic disaccharide units (O, A, iA, B, iB, C, iC, D, iD, E, and iE) (Fig. 1). Different combinations and sequential arrangements of distinct disaccharide units constitute the structural basis for the wide range of biological activities of CS/DS. For example, CS chains containing C, D and E units play distinct roles in bone development [6, 7], neuritogenesis [8, 9], and viral infections , respectively. DS mainly containing iA units regulates the formation of collagen bundles in skin and bones [11-13]. The proportion of E units in CS chains is increased in ovarian and pancreatic cancers, resulting in changes in neoplastic proliferation and cell motility through control of the signaling of vascular endothelial growth factor and the cleavage of CD44, respectively [14-16]. In addition, higher expression of disulfated E units (Fig. 1) has been observed on CS chains at the surface of Lewis lung carcinoma (LLC) cells with high metastatic potential than in those with low metastatic potential, and such CS chains are involved in pulmonary metastasis in an experimental mouse model . We have recently identified receptor for advanced glycation end-products (RAGE), which is a member of the immunoglobulin superfamily and predominantly expressed in the lung, as the receptor molecule at the surface of LLC cells during pulmonary metastasis . This review will focus on recent advances in the study of pulmonary metastasis involving CS chains containing E units [GlcUA-GalNAc(4S,6S)] and RAGE.
CS/DS chains produced by metastatic tumor cells
In the tumor microenvironment, drastic alterations such as glycosylation, upregulation or downregulation of the expression of PGs, extracellular matrix (ECM) proteins, and proteases, and remodeling of the ECM and cell surface result in various behavioral characteristics of neoplastic cells, such as cell adhesion, motility, proliferation, responsiveness through receptor activation or inactivation, and invasive activity [4, 5, 19-22]. These alterations allow tumor cells to vascularize, invade, circulate in the blood, metastasize, and proliferate at metastatic lesions [23-25].
It is well known that HS PGs play important roles in tumor proliferation, metastasis, invasion, adhesion, and angiogenesis [4, 5]. On the other hand, levels of CS/DS PGs are often increased in the stroma, and it has long been thought that the ECM or architectural components of the stroma do not have much biological significance in tumors . However, accumulating evidence indicates that CS/DS PGs, in addition to HS PGs, contribute to the functions and phenotypes of tumor cells as effectors or modulator macromolecules [14-18, 27-29]. The biosynthesis of CS/DS PGs could be upregulated in both tumor stroma and neoplastic cells, resulting in the abundant accumulation of these components in the tumor stroma adjacent to neoplastic cells [30-34]. These observations suggest that CS/DS PGs affect not only tumor progression but also noncancerous host cells such as fibroblasts. For example, CS PG secreted from melanoma stimulates invasion through the regulation of matrix metaroprotease , and myoblasts, under fibrotic and malignant conditions, control tumor invasion and angiogenesis .
Furthermore, distinct sulfation patterns have also been found in the stroma. For instance, CS/DS chains from Engelbreth–Holm–Swarm mouse tumors consist of A units (51%), C units (22%), E units (15%), and O units (11%) . In addition, E units are more abundant in ovarian carcinomas than in normal tissues . These observations suggest that CS chains containing E units are key regulators in tumor development. In fact, the expression of mRNA for GalNAc-4-sulfate-6-O-sulfotransferase (GalNAc4S-6ST), which is responsible for the biosynthesis of E units in CS chains, is upregulated in ovarian and colorectal carcinomas [14, 38]. Interestingly, CS-E binds to vascular endothelial growth factor  in addition to heparin-binding proteins such as fibroblast growth factors, heparin-binding epidermal growth factor-like growth factor, midkine, and pleiotrophin , and colocalizes with blood vessels in ovarian cancer, indicating that CS chains containing E units are involved in tumor angiogenesis. In pancreatic tumor cells, the cleavage of CD44 promotes tumor cell migration . CS-E is expressed in pancreatic tumors and enhances CD44 cleavage, resulting in the alteration of tumor cell motility, suggesting that CS-E modulates cell migration by interacting with the CD44 of tumor cells .
DS epimerase-1, which converts βGlcUA into αIdoUA by epimerizing the C-5 position of GlcUA residues after the formation of chondroitin as a precursor backbone, is highly expressed in esophagus squamous cell carcinoma, and the IdoUA content of DS and CS/DS hybrid chains from the carcinoma are consequently increased . The migration and invasion of squamous cell carcinomas are dependent on the amount of IdoUA, which interacts with hepatocyte growth factor and extracellular signal-related kinase 1/2 signaling . Taken together, the above findings show that the production of CS/DS hybrid chains is strongly enhanced by cancerous cells, resulting in tumorigenesis by providing alterations in cell behavior. Hence, the development of inhibitors of the biosynthesis of CS/DS chains may lead to a new anticancer treatment.
Tumor metastasis involved in CS/DS PGs
Tumor metastasis is a complex multiprocess involving crosstalk with the stroma, migration, invasion, circulation, arrest in distant capillary beds, extravasation, angiogenesis, and multiplication in the target organ [24, 25, 42]. LLC cell lines isolated from the lungs of a C57BL mouse are widely utilized to explore experimental pulmonary metastasis . The CS PG versican accumulates in LLC cells, and functions as a macrophage activator through Toll-like receptor-2, and its coreceptors Toll-like receptor-6 and CD14 . Activation of the Toll-like receptor complexes and induction of the secretion of tumor necrosis factor-α by myeloid cells result in an enhancement of the metastatic growth of LLC, suggesting that the tumor cells usurp constituents of the host innate immune system, resulting in an inflammatory microenvironment favoring metastatic growth . Intriguingly, the CS side chains on versican contain E units , and a higher proportion of E units is detected in the CS chains on LLC cells with high metastatic potential than in those on LLC cells with low metastatic potential . These observations are also supported by gene expression studies, namely quantitative real-time PCR, which revealed the upregulation of GalNAc4S-6ST, which is responsible for the synthesis of E units in LLC cells, as compared with cells with low metastatic potential . The potential of LLC cells to metastasize to the lungs was efficiently suppressed by the removal of CS side chains on PGs at tumor cell surfaces with chondroitinase ABC, the masking of E units on CS chains with the phage display antibody against CS-E (GD3G7), or pretreatment with CS-E infused through a tail vein in mice , implying that CS chains containing E units play a key role in the metastasis of tumor cells to the lung. Most recently, the stable downregulation of GalNAc4S-6ST in LLC cells was shown to result in a reduction in the proportion of E units, and to markedly inhibit the colonization of the lungs by inoculated LLC cells .
RAGE is a functional receptor for tumor CS chains containing E units involved in pulmonary metastasis
CS-E chains strongly bind to a heparin-binding neurotrophic factor, midkine, and an adhesion molecule, P-selectin [39, 45, 47]. Furthermore, midkine-mediated neuronal cell adhesion is inhibited by CS-E , and CS chains on CSPG4 on breast cancer cells function as P-selectin ligands [48, 49]. These findings imply that specific receptor(s) interacting with CS chains with E units expressed at the surface of tumor cells might exist in the vascular endothelial cells of mouse lungs. Recently, our biochemical and proteomic approaches identified RAGE, which is predominantly expressed in the lung, as the receptor molecule for CS chains containing E units expressed at LLC cell surfaces during pulmonary metastasis . RAGE is a member of the immunoglobulin superfamily originally isolated on the basis of its ability to bind to advanced glycation end-products, which are adducts formed by glycoxidation that accumulate in patients with disorders such as diabetes [50-52]. RAGE strongly binds to CS-E, and moderately interacts with DS and CS-D, which contain predominantly IdoUA-GalNAc(4S) (iA units) and GlcUA-GalNAc(6S)/GlcUA(2S)-GalNAc(6S) (C units/D units), respectively, based on the results from a surface plasmon resonance experiment performed in vitro  (Fig. 2). Furthermore, CS-A and CS-C consisting of GlcUA-GalNAc(4S) and GlcUA-GalNAc(6S), respectively, showed weak but appreciable interactions with RAGE. These observations indicate that RAGE recognizes sulfation patterns or sequences in CS chains, perhaps in addition to IdoUA-containing structures. Interestingly, HS and heparin also showed high affinity for RAGE  (Fig. 2). Xu et al.  independently demonstrated that HS regulates the signaling of high-mobility group protein B1, which is a ligand of RAGE, through the formation of a complex of RAGE and HS. In addition, Rao et al.  reported that HS binds RAGE. Thus, HS, in addition to CS/DS, might play roles in RAGE-mediated cell signaling, tumor metastasis, and other biological phenomena.
RAGE has been crystallized, and X-ray crystallography has revealed an elongated molecular shape with a large positively charged region on the surface with direct implications for the binding of negatively charged ligands [55, 56]. In fact, CS-E and HS interact with peptides derived from the basic amino acid region . Interestingly, RAGE recognizes the E unit-containing decasaccharides , which inhibits the pulmonary metastasis of LLC cells . Moreover, a phage display single-chain antibody GD3G7, which recognizes a series of decasaccharide sequences  derived from CS-E of squid cartilage , strongly suppresses RAGE-mediated pulmonary metastasis of LLC cells . These observations suggest that the interaction between RAGE and CS-E appears to require at least a nonasaccharide or decasaccharide length with E unit-containing CS structures such as those shown in Table 1. Note that the antibody GD3G7 interacts also with DS-E rich in iE units (Fig. 1) and .
Table 1. A series of decasaccharide sequences that are possibly recognized by RAGE. A purified CS-E polysaccharide preparation (super special grade; Seikagaku Corp., Tokyo) was digested with testicular hyaluronidase, and fractionated by gel filtration . The obtained octasaccharide and decasaccharide fractions (shown in the far left column) were further fractionated by anion exchange HPLC into 20 and 23 distinct subfractions, respectively. Saccharide components contained in these subfractions were sequenced by digestion with chondroitinases ABC and ACII in conjunction with anion exchange HPLC, as previously reported , and are shown in the far right column. E, A and C stand for GlcUA-GalNAc(4S,6S), GlcUA-GalNAc(4S), and GlcUA-GalNAc(6S), respectively
a Plus (+) and minus (−) indicate positive and negative reactivities towards GD3G7 . b Plus (+) and minus (−) indicate positive and negative antimetastatic activities of size-defined oligosaccharides of CS-E . c Plus (+) and minus (−) indicate positive and negative reactivities of recombinant RAGE .
To verify that the receptor for CS-E, RAGE, expressed in mouse lungs is involved in the initial process of metastasis, including tumor cell adhesion to vascular endothelial cells of the lung, the antibody against RAGE was used for antimetastasis assays for LLC and B16 melanoma cells, which express CS chains containing O, A and C units as the major disaccharides, with E units below the detection limit . However, B16 melanoma cells expressed 1.5 times more DS chains than LLC cells . DS chains also interact with RAGE (Fig. 2). A marked reduction in pulmonary metastasis was observed  (Fig. 3), suggesting that GAG chains, including CS-E, DS and CS/DS hybrid chains on not only mouse LLC cells but also on B16 melanoma cells, and their presumed receptor, RAGE, expressed in mouse lungs are crucial for the pulmonary metastasis of these tumor cells (Fig. 4). In fact, in our preliminary study, no tumor metastasis was observed in the RAGE-knockout mice (S. Mizumoto, J. Takahashi, K. Sugahara, unpublished result).
Recently, Staquicini et al.  have identified RAGE and leukocyte proteinase-3 as molecular partners by screening for vascular ligand receptors in cancer patients. RAGE is upregulated on human prostate cancer cells, and forms a complex with leukocyte proteinase-3 that is relevant to bone marrow-specific tumorigenesis and metastasis, and thereby mediates the homing of metastatic prostate cancer cells to the bone marrow . Thus, RAGE-mediated tumor metastasis may be dependent on the expression of RAGE in the tumor cells and vasculature, and the interaction with a variety of ligands in a tissue-specific or cell type-specific manner.
The recent observations described above suggest that CS oligosaccharides and/or polysaccharides containing E units, HS chains, or their mimetics, and an antibody against CS-E such as GD3G7 have great potential for the development of GAG-based anticancer agents (Fig. 4). Comprehensive or concise reviews have focused on key roles of GAGs that have been demonstrated recently in cancer cell biology [60-62]. Furthermore, RAGE plays an essential role in numerous pathological conditions, such as cancers, inflammatory diseases, diabetes, fibrosis, acute respiratory distress syndrome, inflammation, osteoclast maturation, and physiological neurite outgrowth, through binding to its respective ligands [50, 51, 63, 64]. Further studies on the molecular mechanisms underlying pathological conditions involving RAGE, CS/DS and/or HS PGs will provide insights into new therapeutic approaches for not only lung metastasis but also various other diseases.
This work was supported in part by Grant-in-aid for Scientific Research (B) 23390016 (to K. Sugahara) and Grant-in-aid for Young Scientists (B) 23790066 (to S. Mizumoto) from the Japan Society for the Promotion of Science, Japan (JSPS), the Matching Program for Innovations in Future Drug Discovery and Medical Care (to K. Sugahara) from The Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT), and a Grant-in-aid for Encouragement from The Akiyama Life Science Foundation (to S. Mizumoto).