Targeting stromal cell Syndecan-2 reduces breast tumour growth, metastasis and limits immune evasion.

Tumour stromal cells support tumourigenesis. We report that Syndecan-2 (SDC2) is expressed on a nonepithelial, nonhaematopoietic, nonendothelial stromal cell population within breast cancer tissue. In vitro, syndecan-2 modulated TGFß signalling (SMAD7, PAI-1), migration and immunosuppression of patient-derived tumour-associated stromal cells (TASCs). In an orthotopic immunocompromised breast cancer model, overexpression of syndecan-2 in TASCs significantly enhanced TGFß signalling (SMAD7, PAI-1), tumour growth and metastasis, whereas reducing levels of SDC2 in TASCs attenuated TGFß signalling (SMAD7, PAI-1, CXCR4), tumour growth and metastasis. To explore the potential for therapeutic application, a syndecan-2-peptide was generated that inhibited the migratory and immunosuppressive properties of TASCs in association with reduced expression of TGFß-regulated immunosuppressive genes, such as CXCR4 and PD-L1. Moreover, using an orthotopic syngeneic breast cancer model, overexpression of syndecan-2-peptide in TASCs reduced tumour growth and immunosuppression within the TME. These data provide evidence that targeting stromal syndecan-2 within the TME inhibits tumour growth and metastasis due to decreased TGFß signalling and increased immune control.

Syndecan-2 in colorectal cancer plays oncogenic role via epithelial-mesenchymal transition and MAPK pathway.

PURPOSE: In this study, we aimed to elucidate the biological roles of Syndecan-2 (SDC2) in colorectal cancer (CRC), thereby further understanding its clinical role. METHODS: The expression of SDC2 was assessed by qRT-PCR and Western blot analysis. To understand the potential biological role of SDC2, we also explored the correlation between its expression level and clinicopathologic parameters. By using MTT, plate colony formation assay, Transwell invasion assays, and flow cytometry in vitro, the biological impact of SDC2 on CRC cell proliferation, migration, invasion, and apoptosis. In addition, the related signaling pathways were investigated. RESULTS: SDC2 expression was significantly upregulated in CRC tissues. The expression of SDC2 was highly associated with four parameters, i.e., stage (P < 0.01), vascular invasion (P = 0.0045), lymph node metastasis (P=0.0018), and distant metastasis (P = 0.0019). Knockdown of SDC2 significantly reduced proliferation, migration, and invasion of HCT116 and SW480 cells, and induced cell apoptosis. Moreover, SDC2 promoted epithelial-mesenchymal transition (EMT) in CRC cells, whereas the ratio of p-MEK/MEK and p-ERK/ERK markedly reduced after depleting SDC2. CONCLUSION: During CRC development, overexpression of SDC2 plays a carcinogenic role in CRC. Therapeutic solutions targeting SDC2 may provide potential insights into CRC prevention and treatment.

N-terminal syndecan-2 domain selectively enhances 6-O heparan sulfate chains sulfation and promotes VEGFA165-dependent neovascularization.

The proteoglycan Syndecan-2 (Sdc2) has been implicated in regulation of cytoskeleton organization, integrin signaling and developmental angiogenesis in zebrafish. Here we report that mice with global and inducible endothelial-specific deletion of Sdc2 display marked angiogenic and arteriogenic defects and impaired VEGFA165 signaling. No such abnormalities are observed in mice with deletion of the closely related Syndecan-4 (Sdc4) gene. These differences are due to a significantly higher 6-O sulfation level in Sdc2 versus Sdc4 heparan sulfate (HS) chains, leading to an increase in VEGFA165 binding sites and formation of a ternary Sdc2-VEGFA165-VEGFR2 complex which enhances VEGFR2 activation. The increased Sdc2 HS chains 6-O sulfation is driven by a specific N-terminal domain sequence; the insertion of this sequence in Sdc4 N-terminal domain increases 6-O sulfation of its HS chains and promotes Sdc2-VEGFA165-VEGFR2 complex formation. This demonstrates the existence of core protein-determined HS sulfation patterns that regulate specific biological activities.

Syndecan-2 regulates melanin synthesis via protein kinase C ßII-mediated tyrosinase activation.

Syndecan-2, a transmembrane heparan sulfate proteoglycan that is highly expressed in melanoma cells, regulates melanoma cell functions (e.g. migration). Since melanoma is a malignant tumor of melanocytes, which largely function to synthesize melanin, we investigated the possible involvement of syndecan-2 in melanogenesis. Syndecan-2 expression was increased in human skin melanoma tissues compared with normal skin. In both mouse and human melanoma cells, siRNA-mediated knockdown of syndecan-2 was associated with reduced melanin synthesis, whereas overexpression of syndecan-2 increased melanin synthesis. Similar effects were also detected in human primary epidermal melanocytes. Syndecan-2 expression did not affect the expression of tyrosinase, a key enzyme in melanin synthesis, but instead enhanced the enzymatic activity of tyrosinase by increasing the membrane and melanosome localization of its regulator, protein kinase CßII. Furthermore, UVB caused increased syndecan-2 expression, and this up-regulation of syndecan-2 was required for UVB-induced melanin synthesis. Taken together, these data suggest that syndecan-2 regulates melanin synthesis and could be a potential therapeutic target for treating melanin-associated diseases.

Syndecan-2 exerts antifibrotic effects by promoting caveolin-1-mediated transforming growth factor-ß receptor I internalization and inhibiting transforming growth factor-ß1 signaling.

RATIONALE: Alveolar transforming growth factor (TGF)-ß1 signaling and expression of TGF-ß1 target genes are increased in patients with idiopathic pulmonary fibrosis (IPF) and in animal models of pulmonary fibrosis. Internalization and degradation of TGF-ß receptor TßRI inhibits TGF-ß signaling and could attenuate development of experimental lung fibrosis. OBJECTIVES: To demonstrate that after experimental lung injury, human syndecan-2 confers antifibrotic effects by inhibiting TGF-ß1 signaling in alveolar epithelial cells. METHODS: Microarray assays were performed to identify genes differentially expressed in alveolar macrophages of patients with IPF versus control subjects. Transgenic mice that constitutively overexpress human syndecan-2 in macrophages were developed to test the antifibrotic properties of syndecan-2. In vitro assays were performed to determine syndecan-2-dependent changes in epithelial cell TGF-ß1 signaling, TGF-ß1, and TßRI internalization and apoptosis. Wild-type mice were treated with recombinant human syndecan-2 during the fibrotic phase of bleomycin-induced lung injury. MEASUREMENTS AND MAIN RESULTS: We observed significant increases in alveolar macrophage syndecan-2 levels in patients with IPF. Macrophage-specific overexpression of human syndecan-2 in transgenic mice conferred antifibrotic effects after lung injury by inhibiting TGF-ß1 signaling and downstream expression of TGF-ß1 target genes, reducing extracellular matrix production and alveolar epithelial cell apoptosis. In vitro, syndecan-2 promoted caveolin-1-dependent internalization of TGF-ß1 and TßRI in alveolar epithelial cells, which inhibited TGF-ß1 signaling and epithelial cell apoptosis. Therapeutic administration of human syndecan-2 abrogated lung fibrosis in mice. CONCLUSIONS: Alveolar macrophage syndecan-2 exerts antifibrotic effects by promoting caveolin-1-dependent TGF-ß1 and TßRI internalization and inhibiting TGF-ß1 signaling in alveolar epithelial cells. Hence, molecules that facilitate TßRI degradation via endocytosis represent potential therapies for pulmonary fibrosis.

Syndecan-2 is upregulated in colorectal cancer cells through interactions with extracellular matrix produced by stromal fibroblasts.

BACKGROUND: The extracellular matrix (ECM) influences the structure, viability and functions of cells and tissues. Recent evidence indicates that tumor cells and stromal cells interact through direct cell-cell contact, the production of ECM components and the secretion of growth factors. Syndecans are a family of transmembrane heparan sulfate proteoglycans that are involved in cell adhesion, motility, proliferation and differentiation. Syndecan-2 has been found to be highly expressed in colorectal cancer cell lines and appears to be critical for cancer cell behavior. We have examined the effect of stromal fibroblast-produced ECM on the production of proteoglycans by colorectal cancer cell lines. RESULTS: Our results showed that in a highly metastatic colorectal cancer cell line, HCT-116, syndecan-2 expression is enhanced by fibroblast ECM, while the expression of other syndecans decreased. Of the various components of the stromal ECM, fibronectin was the most important in stimulating the increase in syndecan-2 expression. The co-localization of syndecan-2 and fibronectin suggests that these two molecules are involved in the adhesion of HCT-116 cells to the ECM. Additionally, we demonstrated an increase in the expression of integrins alpha-2 and beta-1, in addition to an increase in the expression of phospho-FAK in the presence of fibroblast ECM. Furthermore, blocking syndecan-2 with a specific antibody resulted in a decrease in cell adhesion, migration, and organization of actin filaments. CONCLUSIONS: Overall, these results show that interactions between cancer cells and stromal ECM proteins induce significant changes in the behavior of cancer cells. In particular, a shift from the expression of anti-tumorigenic syndecans to the tumorigenic syndecan-2 may have implications in the migratory behavior of highly metastatic tumor cells.

Syndecan 4 supports bone fracture repair, but not fetal skeletal development, in mice.

OBJECTIVE: Syndecan 4, a heparan sulfate proteoglycan, has been associated with osteoarthritis. The present study was undertaken to analyze the functional role of syndecan 4 in endochondral ossification of mouse embryos and in adult fracture repair, which, like osteoarthritis, involves an inflammatory component. METHODS: Sdc4 promoter activity was analyzed in Sdc4(-/-) lacZ-knockin mice, using ß-galactosidase staining. Endochondral ossification in embryos from embryonic day 16.5 was assessed by histologic and immunohistologic staining. Bone fracture repair was analyzed in femora of adult mice on days 7 and 14 postfracture. To evaluate Sdc2 and Sdc4 gene expression with and without tumor necrosis factor α (TNFα) and Wnt-3a stimulation, quantitative real-time polymerase chain reaction was performed. RESULTS: In Sdc4(-/-) lacZ-knockin animals, syndecan 4 promoter activity was detectable at all stages of chondrocyte differentiation, and Sdc4 deficiency inhibited chondrocyte proliferation. Aggrecan turnover in the uncalcified cartilage of the epiphysis was decreased transiently in vivo, but this did not lead to a growth phenotype at birth. In contrast, among adult mice, fracture healing was markedly delayed in Sdc4(-/-) animals and was accompanied by increased callus formation. Blocking of inflammation via anti-TNFα treatment during fracture healing reduced these changes in Sdc4(-/-) mice to levels observed in wild-type controls. We analyzed the differences between the mild embryonic and the severe adult phenotype, and found a compensatory up-regulation of syndecan 2 in the developing cartilage of Sdc4(-/-) mice that was absent in adult tissue. Stimulation of chondrocytes with Wnt-3a in vitro led to increased expression of syndecan 2, while stimulation with TNFα resulted in up-regulation of syndecan 4 but decreased expression of syndecan 2. TNFα stimulation reduced syndecan 2 expression and increased syndecan 4 expression even in the presence of Wnt-3a, suggesting that inflammation has a strong effect on the regulation of syndecan expression. CONCLUSION: Our results demonstrate that syndecan 4 is functionally involved in endochondral ossification and that its loss impairs fracture healing, due to inhibition of compensatory mechanisms under inflammatory conditions.

Calpain-6 is an endothelin-1 signaling dependent protective factor in chemoresistant osteosarcoma.

Bone tumors strongly influence normal tissues and stimulate bone cells for the production of cytokines supporting proliferation and abnormal survival in cancer cells. We previously reported that the proteoglycan syndecan-2 controls the activity of various cytokines and growth factors and also modulates apoptosis and response to cytotoxic agents in osteosarcoma cell lines. Here, we show that syndecan-2 has a stronger tumor suppressor activity in vivo. We identify calpain-6 as a target gene downregulated by syndecan-2 in cells and in vivo. We demonstrate that calpain-6 expression in osteosarcoma cells depends on endothelin-1, a mediator of the tumor progression in bone. Syndecan-2 overexpression alters ERK1/2, PI3K/AKT and NFκB pathways that are calpain-6-promoting signals downstream of endothelin-1. Immunohistochemical analysis shows that calpain-6 is expressed in human bone tumors and metastases. A high expression of calpain-6 was specially found in recurrent osteosarcoma. Moreover, calpain-6 levels in primary tumors were inversely related to the response to chemotherapy. Consistently, calpain-6 was increased by doxorubicin and was found to be expressed at higher levels in doxorubicin-resistant U2OS osteosarcoma-derived cells as compared to responsive cells. Inhibition of calpain-6 with shRNA resulted in decreased proliferation, increased spontaneous apoptosis and increased sensitivity to doxorubicin and also methotrexate in responsive and resistant osteosarcoma cells. Taken together, our data show that syndecan-2 exerts its pro-apoptotic function through modulation of the endothelin-1/NFκB signaling and through downregulation of calpain-6, a protective factor that contributes to abnormal cell survival. Thus, this study identifies calpain-6 as a new possible therapeutic target in chemoresistant osteosarcoma.

The expression of syndecan-1 and -2 is associated with Gleason score and epithelial-mesenchymal transition markers, E-cadherin and beta-catenin, in prostate cancer.

The epithelial-mesenchymal transition (EMT) is considered a key step in tumor progression, where the invasive cancer cells change from epithelial to mesenchymal phenotype. During this process, a decrease or loss in adhesion molecules expression and an increase in migration molecules expression are observed. The aim of this work was to determine the expression and cellular distribution of syndecan-1 and -2 (migration molecules) and E-cadherin and beta-catenin (adhesion molecules) in different stages of prostate cancer progression. A quantitative immunohistochemical study of these molecules was carried out in tissue samples from benign prostatic hyperplasia and prostate carcinoma, with low and high Gleason score, obtained from biopsies archives of the Clinic Hospital of the University of Chile and Dipreca Hospital. Polyclonal specific antibodies and amplification system of estreptavidin-biotin peroxidase and diaminobenzidine were used. Syndecan-1 was uniformly expressed in basolateral membranes of normal epithelium, changing to a granular cytoplasmatic expression pattern in carcinomas. Syndecan-2 was observed mainly in a cytoplasmatic granular pattern, with high immunostaining intensity in areas of low Gleason score. E-cadherin was detected in basolateral membrane of normal epithelia showing decreased expression in high Gleason score samples. beta-Catenin was found in cell membranes of normal epithelia changing its distribution toward the nucleus and cytoplasm in carcinoma samples. We concluded that changes in expression and cell distribution of E-cadherin and beta-catenin correlated with the progression degree of prostate adenocarcinoma, suggesting a role of these molecules as markers of progression and prognosis. Furthermore, changes in the pattern expression of syndecan-1 and -2 indicate that both molecules may be involved in the EMT and tumor progression of prostate cancer.

Is syndecan-2 a key angiogenic element?

Angiogenesis has been extensively related with the development of many different diseases, from tumor progression to cardiovascular disorders. In our last article, we demonstrated that syndecan-2, the most abundant heparan sulfate proteoglycan expressed by human microvascular endothelial cells, is regulated by proangiogenic factors and plays an important role in most of the cellular events that take place in neovascularization processes. Here, we also reported its involvement in the reorganization of the cytoskeleton and propose a key role for this proteoglycan as an adaptor protein, able to work in cooperation with the integrins in the formation of new blood vessels.

Syndecan-2 downregulation impairs angiogenesis in human microvascular endothelial cells.

The formation of new blood vessels, or angiogenesis, is a necessary process during development but also for tumour growth and other pathologies. It is promoted by different growth factors that stimulate endothelial cells to proliferate, migrate, and generate new tubular structures. Syndecans, transmembrane heparan sulphate proteoglycans, bind such growth factors through their glycosaminoglycan chains and could transduce the signal to the cytoskeleton, thus regulating cell behaviour. We demonstrated that syndecan-2, the major syndecan expressed by human microvascular endothelial cells, is regulated by growth factors and extracellular matrix proteins, in both bidimensional and tridimensional culture conditions. The role of syndecan-2 in "in vitro" tumour angiogenesis was also examined by inhibiting its core protein expression with antisense phosphorothioate oligonucleotides. Downregulation of syndecan-2 reduces spreading and adhesion of endothelial cells, enhances their migration, but also impairs the formation of capillary-like structures. These results suggest that syndecan-2 has an important function in some of the necessary steps that make up the angiogenic process. We therefore propose a pivotal role of this heparan sulphate proteoglycan in the formation of new blood vessels.

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.

Syndecan-2 induces filopodia and dendritic spine formation via the neurofibromin-PKA-Ena/VASP pathway.

Syndecan-2 induced filopodia before spinogenesis; therefore, filopodia formation was used here as a model to study the early downstream signaling of syndecan-2 that leads to spinogenesis. Screening using kinase inhibitors indicated that protein kinase A (PKA) is required for syndecan-2-induced filopodia formation in both human embryonic kidney cells and hippocampal neurons. Because neurofibromin, a syndecan-2-binding partner, activates the cyclic adenosine monophosphate pathway, the role of neurofibromin in syndecan-2-induced filopodia formation was investigated by deletion mutant analysis, RNA interference, and dominant-negative mutant. The results showed that neurofibromin mediates the syndecan-2 signal to PKA. Among actin-associated proteins, Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) were predicted as PKA effectors downstream of syndecan-2, as Ena/VASP, which is activated by PKA, induces actin polymerization. Indeed, when the activities of Ena/VASP were blocked, syndecan-2 no longer induced filopodia formation. Finally, in addition to filopodia formation, neurofibromin and Ena/VASP contributed to spinogenesis. This study reveals a novel signaling pathway in which syndecan-2 activates PKA via neurofibromin and PKA consequently phosphorylates Ena/VASP, promoting filopodia and spine formation.

Syndecan-2 expression increases serum-withdrawal-induced apoptosis, mediated by re-distribution of Fas into lipid rafts, in stably transfected Swiss 3T3 cells.

To examine the function of syndecan-2, one of the most abundant heparan sulfate proteoglycans in fibroblasts, we obtained stably transfected Swiss 3T3 clones. We examined the effects of stable syndecan-2 overexpression on programmed cell death, finding that syndecan-2 transfected cells were more sensitive to apoptosis induced by serum-withdrawal than control cells. In addition, overexpression of syndecan-2 correlates with increased membrane levels of the Fas/CD95 receptor, suggesting that the increased serum-withdrawal apoptosis observed in Swiss 3T3 cells might be Fas receptor-dependent. Differences in Fas membrane levels between both control and syndecan-2 transfected cells result from a redistribution of the Fas receptor. Our data clearly demonstrate that increased Fas levels are primarily related to lipid rafts and that this increase is a key factor in Fas/CD95-mediated apoptosis. Moreover, disruption of lipid rafts with methyl-beta-cyclodextrin or filipin significantly reduced apoptosis in response to serum withdrawal. The differences in Fas/CD95 membrane distribution could explain why syndecan-2 transfected cells have a higher susceptibility to serum-withdrawal-induced apoptosis.