Hyaluronan deficiency in tumor stroma impairs macrophage trafficking and tumor neovascularization.

Despite the importance of stromal cells in tumor progression, our overall understanding of the molecular signals that regulate the complex cellular interactions within tumor stroma is limited. Here, we provide multiple lines of evidence that tumor-associated macrophages (TAM) preferentially traffic to stromal areas formed within tumors in a manner dependent on a hyaluronan (HA)-rich tumor microenvironment. To address the role of stroma-derived HA in macrophage recruitment, we disrupted the HA synthase 2 (Has2) gene in stromal fibroblasts using conditional gene targeting. The Has2 null fibroblasts showed severe impairment in recruiting macrophages when inoculated with tumor cells into nude mice, which shows the contribution of stroma-derived HA in intratumoral macrophage mobilization. Furthermore, a deficiency in stromal HA attenuated tumor angiogenesis and lymphangiogenesis concomitantly with impaired macrophage recruitment. Taken together, our results suggest that stromal HA serves as a microenvironmental signal for the recruitment of TAMs, which are key regulatory cells involved in tumor neovascularization.

Involvement of highly sulfated chondroitin sulfate in the metastasis of the Lewis lung carcinoma cells.

The altered expression of cell surface chondroitin sulfate (CS) and dermatan sulfate (DS) in cancer cells has been demonstrated to play a key role in malignant transformation and tumor metastasis. However, the functional highly sulfated structures in CS/DS chains and their involvement in the process have not been well documented. In the present study, a structural analysis of CS/DS from two mouse Lewis lung carcinoma (3LL)-derived cell lines with different metastatic potentials revealed a higher proportion of Delta(4,5)HexUA-GalNAc(4,6-O-disulfate) generated from E-units (GlcUA-GalNAc(4, 6-O-disulfate)) in highly metastatic LM66-H11 cells than in low metastatic P29 cells, although much less CS/DS is expressed by LM66-H11 than P29 cells. This key finding prompted us to study the role of CS-E-like structures in experimental lung metastasis. The metastasis of LM66-H11 cells to lungs was effectively inhibited by enzymatic removal of tumor cell surface CS or by preadministration of CS-E rich in E-units in a dose-dependent manner. In addition, immunocytochemical analysis showed that LM66-H11 rather than P29 cells expressed more strongly the CS-E epitope, which was specifically recognized by the phage display antibody GD3G7. More importantly, this antibody and a CS-E decasaccharide fraction, the minimal structure recognized by GD3G7, strongly inhibited the metastasis of LM66-H11 cells probably by modifying the proliferative and invading behavior of the metastatic tumor cells. These results suggest that the E-unit-containing epitopes are involved in the metastatic process and a potential target for the diagnosis and treatment of malignant tumors.

Matrix metalloproteinase-9 associated with heparan sulphate chains of GPI-anchored cell surface proteoglycans mediates motility of murine colon adenocarcinoma cells.

Using murine colon adenocarcinoma-derived clones with different metastatic potentials, the cellular localization of matrix metalloporteinase-9 (MMP-9) and its role in the cell motility were examined. Highly metastatic LuM1 clone aggressively invaded into adjacent tissue in vivo, but low metastatic NM11 clone did not. As compared with the NM11 clone, the LuM1 clone expressed and secreted a remarkably large amount of MMP-9, and exhibited higher abilities of cell migration and invasion in vitro, which were suppressed by MMP-2/MMP-9 inhibitor IV. MMP-9, exhibiting high affinity to heparin, was demonstrated to be condensed on tips of cellular podia. Treatment of the cells with heparitinase-I or heparin resulted in release of MMP-9 from the cell surface, which caused concomitant suppression of their motility to a similar level to that with the MMP inhibitor. Immunoprecipitation of a LuM1 cell lysate with an anti-MMP-9 antibody resulted in co-precipitation of phosphatidylinositol-specific phospholipase C-susceptible heparan sulphate proteoglycans having 66 and 64 kDa core proteins. Taken together, the present results demonstrate that secreted MMP-9 associates with glypican-like proteoglycans through their heparan sulphate chains, and plays a crucial role in cell motility of LuM1 cells.

Differential expression of syndecan isoforms during mouse incisor amelogenesis.

Syndecans are transmembranous heparan sulfate proteoglycans (HSPGs) with covalently attached glycosaminoglycan side-chains located on the cell surface. The mammalian syndecan family is composed of four types of syndecans (syndecan-1 to -4). Syndecans interact with the intracellular cytoskeleton through the cytoplasmic domains of their core proteins and membrane proteins, extracellular enzymes, growth factors, and matrix components, through their heparan-sulfate chains, to regulate developmental processes.Here, as a first step to assess the possible roles of syndecan proteins in amelogenesis, we examined the expression patterns of all syndecan isoforms in continuously growing mouse incisors, in which we can overview major differentiation stages of amelogenesis at a glance. Understanding the expression domain of each syndecan isoform during specific developmental stages seems useful for investigating their physiological roles in amelogenesis. Immunohistochemical analysis of syndecan core proteins in the lower incisors from postnatal day 1 mice revealed spatially and temporally specific expression patterns, with syndecan-1 expressed in undifferentiated epithelial and mesenchymal cells, and syndecan-2, -3, and -4 in more differentiated cells. These findings suggest that each syndecan isoform functions distinctly during the amelogenesis of the incisors of mice.

A novel function of syndecan-2, suppression of matrix metalloproteinase-2 activation, which causes suppression of metastasis.

The syndecans comprise a family of cell surface heparan sulfate proteoglycans exhibiting complex biological functions involving the interaction of heparan sulfate side chains with a variety of soluble and insoluble heparin-binding extracellular ligands. Here we demonstrate an inverse correlation between the expression level of syndecan-2 and the metastatic potential of three clones derived from Lewis lung carcinoma 3LL. This correlation was proved to be a causal relationship, because transfection of syndecan-2 into the higher metastatic clone resulted in the suppression of both spontaneous and experimental metastases to the lung. Although the expression levels of matrix metalloproteinase-2 (MMP-2) and its cell surface activators, such as membrane-type 1 matrix metalloproteinase and tissue inhibitor of metalloproteinase-2, were similar regardless of the metastatic potentials of the clones, elevated activation of MMP-2 was observed in the higher metastatic clone. Removal of heparan sulfate from the cell surface of low metastatic cells by treatment with heparitinase-I promoted MMP-2 activation, and transfection of syndecan-2 into highly metastatic cells suppressed MMP-2 activation. Furthermore, transfection of mutated syndecan-2 lacking glycosaminoglycan attachment sites into highly metastatic cells did not have any suppressive effect on MMP-2 activation, suggesting that this suppression was mediated by the heparan sulfate side chains of syndecan-2. Actually, MMP-2 was found to exhibit a strong binding ability to heparin, the dissociation constant value being 62 nM. These results indicate a novel function of syndecan-2, which acts as a suppressor for MMP-2 activation, causing suppression of metastasis in at least the metastatic system used in the present study.

Midkine, a heparin-binding growth factor, produced by the host enhances metastasis of Lewis lung carcinoma cells.

Midkine is a heparin-binding growth factor and is expressed by a number of tumor cells, contributing to their growth both in vitro and in vivo. Spontaneous lung metastasis of Lewis lung carcinoma cells, which did not significantly express MK, was significantly less extensive in mice deficient in the midkine gene than in wild-type mice, when the tumor was subcutaneously grown above the thigh. Midkine strongly enhanced migration of Lewis lung carcinoma cells in vitro. Therefore, midkine is also a host factor enhancing tumor metastasis. Anti-midkine therapy for malignancy may act on midkine produced by both the tumor and host.

Heparan sulphate proteoglycans interact with neurocan and promote neurite outgrowth from cerebellar granule cells.

We found that neurocan, a major brain chondroitin sulphate proteoglycan, interacts with HSPGs (heparan sulphate proteoglycans) such as syndecan-3 and glypican-1. Binding of these HSPGs to neurocan was prevented by treatment of the HSPGs with heparitinases I and II, but not by treatment of neurocan with chondroitinase ABC. Scatchard plot analysis indicated that neurocan has two binding sites for these HSPGs with different affinities. It is known that neurocan in the rodent brain is proteolytically processed with aging into N- and C-terminal fragments. When a mixture of whole neurocan and N- and C-terminal fragments prepared from neonatal mouse brains or recombinant N- and C-terminal fragments was applied to a heparin column, the whole molecule and both the N- and C-terminal fragments bound to heparin. A centrifugation cell adhesion assay indicated that both the N- and C-terminal neurocan fragments could interact with these HSPGs expressed on the cell surface. To examine the biological significance of the HSPG-neurocan interaction, cerebellar granule cells expressing these HSPGs were cultured on the recombinant neurocan substrate. A significant increase in the rate of neurite outgrowth was observed on the wells coated with the C-terminal neurocan fragment, but not with the N-terminal one. Neurite outgrowth-promoting activity was inhibited by pretreatment of neurocan substrate with heparin or the addition of heparitinase I to culture medium. These results suggest that HSPGs such as syndecan-3 and glypican-1 serve as the cell-surface receptor of neurocan, and that the interaction of these HSPGs with neurocan through its C-terminal domain is involved in the promotion of neurite outgrowth.

Inhibition of experimental lung metastases of Lewis lung carcinoma cells by chemically modified heparin with reduced anticoagulant activity.

Heparin, a widely used anticoagulant, is known to have anti-metastatic activity, although the mechanism is not fully understood. In the present study, we investigated the mechanism of this anti-metastatic activity using periodate-oxidized and borohydride-reduced heparin with low anticoagulant activity (LAC heparin). The anticoagulant activity of LAC heparin is markedly reduced to almost the control level in terms of prothrombin time in vitro, and no hemorrhagic complication was observed with injection of LAC heparin into mice in vivo. LAC heparin injected intravenously with Lewis lung carcinoma cells or 10 min before tumor cell injection significantly inhibited, to the same extent as intact heparin and in a dose- and time-dependent manner, the lung colonization that develops after intravenous injection (i.v.) of tumor cells. Flow cytometric analysis revealed that Lewis lung carcinoma cells strongly express heparan sulfate on their surface. Both the LAC heparin and intact heparin inhibited the adhesion and invasion of tumor cells to Matrigel-coated dishes in vitro without significant effect on the tumor cell growth. LAC heparin also significantly diminished tumor cell retention in the lung after i.v. of LacZ gene-tagged Lewis lung carcinoma cells. These results suggest that LAC heparin may prevent tumor cells from attachment to the subendothelial matrix of lung capillaries by competitively inhibiting cell surface heparan sulfate functions and suppress lung colonization.

Cooperation of syndecan-2 and syndecan-4 among cell surface heparan sulfate proteoglycans in the actin cytoskeletal organization of Lewis lung carcinoma cells.

Syndecan-2 cooperates with integrin alpha 5 beta 1 in cell adhesion to a fibronectin substratum and regulates actin cytoskeletal organization in an expression level-dependent manner; Lewis lung carcinoma-derived P29 cells with high expression form stress fibers, whereas the same tumor-derived low expressers, LM66-H11 cells, form cortex actin [Munesue, S., Kusano, Y., Oguri, K., Itano, N., Yoshitomi, Y., Nakanishi, H., Yamashina, I., and Okayama, M. (2002) BIOCHEM: J. 363, 201-209]. In this study we examined the participation of other cell surface heparan sulfate proteoglycans in this signaling. The two clones expressed syndecan-1, -2 and -4, and glypican-1 at similar levels except for syndecan-2. Treatment of cells with phosphatidylinositol-specific phospholipase C or immobilized anti-syndecan-1 antibodies demonstrated that neither glypican-1 nor syndecan-1 was involved in this signaling, indicating that individual cell surface heparan sulfate proteoglycans have functional specificity. Stimulation with immobilized anti-syndecan-2 or -4 antibodies induced stress fiber formation in P29 cells but not in LM66-H11 cells, despite the similar levels of syndecan-4 expression, suggesting that stress fiber formation required a threshold expression level of syndecan-2 acting downstream of syndecan-4. This was confirmed by cells in which syndecan-2 expression was artificially suppressed by antisense mRNA oligonucleotide treatment or elevated by cDNA transfection. This is the first report demonstrating that syndecan-2 and -4 cooperate in situ in actin cytoskeletal organization.

The role of syndecan-2 in regulation of actin-cytoskeletal organization of Lewis lung carcinoma-derived metastatic clones.

Syndecans, a family of transmembrane heparan sulphate proteoglycans, contribute to various biological processes, including adhesion, motility, proliferation, differentiation and morphogenesis. We document here the involvement of syndecan-2 acting alone or co-operatively with integrin alpha5beta1, for regulation of actin-cytoskeletal organization on cell adhesion to fibronectin, using fibronectin-recombinant polypeptides containing the ligands for either or both of these receptors as substrata. Lewis lung carcinoma-derived low-metastatic P29 cells binding to the substrata by both receptors formed actin stress fibres, whereas those binding by syndecan-2 or integrin alpha5beta1 alone formed filopodia or cortex actin. In contrast, higher metastatic LM66-H11 cells formed cortex actin even on substrata containing both ligands. Northern-blot and flow-cytometric analyses revealed that syndecan-2 expression in LM66-H11 cells was significantly lower (1/4.5 in mRNA and 1/8 in cell-surface expression) than in P29 cells, whereas expression levels of integrin alpha5beta1 and other syndecans were similar in both cell types. These results suggest that the failure of LM66-H11 to form stress fibres is due to a lower expression of syndecan-2 than that due to a threshold for its function. This was confirmed by the finding that overexpression of syndecan-2 by transfection of its cDNA into LM66-H11 cells caused the formation of stress fibres on the fibronectin substratum. These in vitro cellular responses of the two clones might reflect their in vivo situation in primary tumours in which P29 cells with a stroma-inducing capacity were immediately surrounded by fibronectin-rich matrix formed by the induced stromal cells, whereas LM66-H11 cells without such capacity were not surrounded by a similar matrix.