Heparanase degrades syndecan-1 and perlecan heparan sulfate: functional implications for tumor cell invasion

Heparanase (HPSE-1) is involved in the degradation of both cell-surface and extracellular matrix (ECM) heparan sulfate (HS) in normal and neoplastic tissues. Degradation of heparan sulfate proteoglycans (HSPG) in mammalian cells is dependent upon the enzymatic activity of HPSE-1, an endo-beta-d-glucuronidase, which cleaves HS using a specific endoglycosidic hydrolysis rather than an eliminase type of action. Elevated HPSE-1 levels are associated with metastatic cancers, directly implicating HPSE-1 in tumor progression. The mechanism of HPSE-1 action to promote tumor progression may involve multiple substrates because HS is present on both cell-surface and ECM proteoglycans. However, the specific targets of HPSE-1 action are not known. Of particular interest is the relationship between HPSE-1 and HSPG, known for their involvement in tumor progression. Syndecan-1, an HSPG, is ubiquitously expressed at the cell surface, and its role in cancer progression may depend upon its degradation. Conversely, another HSPG, perlecan, is an important component of basement membranes and ECM, which can promote invasive behavior. Down-regulation of perlecan expression suppresses the invasive behavior of neoplastic cells in vitro and inhibits tumor growth and angiogenesis in vivo. In this work we demonstrate the following. 1) HPSE-1 cleaves HS present on the cell surface of metastatic melanoma cells. 2) HPSE-1 specifically degrades HS chains of purified syndecan-1 or perlecan HS. 3) Syndecan-1 does not directly inhibit HPSE-1 enzymatic activity. 4) The presence of exogenous syndecan-1 inhibits HPSE-1-mediated invasive behavior of melanoma cells by in vitro chemoinvasion assays. 5) Inhibition of HPSE-1-induced invasion requires syndecan-1 HS chains. These results demonstrate that cell-surface syndecan-1 and ECM perlecan are degradative targets of HPSE-1, and syndecan-1 regulates HPSE-1 biological activity. This suggest that expression of syndecan-1 on the melanoma cell surface and its degradation by HPSE-1 are important determinants in the control of tumor cell invasion and metastasis.     

Epac1 increases migration of endothelial cells and melanoma cells via FGF2‐mediated paracrine signaling

Fibroblast growth factor (FGF2) regulates endothelial and melanoma cell migration. The binding of FGF2 to its receptor requires N‐sulfated heparan sulfate (HS) glycosamine. We have previously reported that Epac1, an exchange protein activated by cAMP, increases N‐sulfation of HS in melanoma. Therefore, we examined whether Epac1 regulates FGF2‐mediated cell–cell communication. Conditioned medium (CM) of melanoma cells with abundant expression of Epac1 increased migration of human umbilical endothelial cells (HUVEC) and melanoma cells with poor expression of Epac1. CM‐induced increase in migration was inhibited by antagonizing FGF2, by the removal of HS and by the knockdown of Epac1. In addition, knockdown of Epac1 suppressed the binding of FGF2 to FGF receptor in HUVEC, and in vivo angiogenesis in melanoma. Furthermore, knockdown of Epac1 reduced N‐sulfation of HS chains attached to perlecan, a major secreted type of HS proteoglycan that mediates the binding of FGF2 to FGF receptor. These data suggested that Epac1 in melanoma cells regulates melanoma progression via the HS–FGF2‐mediated cell–cell communication.     

Soluble Heparan Sulfate Fragments Generated by Heparanase Trigger the Release of Pro-Inflammatory Cytokines through TLR-4

Heparanase is a β-D-endoglucuronidase that cleaves heparan sulfate (HS), facilitating degradation of the extracellular matrix (ECM) and the release of HS-bound biomolecules including cytokines. The remodeling of the ECM by heparanase is important for various physiological and pathological processes, including inflammation, wound healing, tumour angiogenesis and metastasis. Although heparanase has been proposed to facilitate leukocyte migration through degradation of the ECM, its role in inflammation by regulating the expression and release of cytokines has not been fully defined. In this study, the role of heparanase in regulating the expression and release of cytokines from human and murine immune cells was examined. Human peripheral blood mononuclear cells treated ex vivo with heparanase resulted in the release of a range of pro-inflammatory cytokines including IL-1β, IL-6, IL-8, IL-10 and TNF. In addition, mouse splenocytes treated ex vivo with heparanase resulted in the release of IL-6, MCP-1 and TNF. A similar pattern of cytokine release was also observed when cells were treated with soluble HS. Furthermore, heparanase-induced cytokine release was abolished by enzymatic-inhibitors of heparanase, suggesting this process is mediated via the enzymatic release of cell surface HS fragments. As soluble HS can signal through the Toll-like receptor (TLR) pathway, heparanase may promote the upregulation of cytokines through the generation of heparanase-cleaved fragments of HS. In support of this hypothesis, mouse spleen cells lacking the key TLR adaptor molecule MyD88 demonstrated an abolition of cytokine release after heparanase stimulation. Furthermore, TLR4-deficient spleen cells showed reduced cytokine release in response to heparanase treatment, suggesting that TLR4 is involved in this response. Consistent with these observations, the pathway involved in cytokine upregulation was identified as being NF-κB-dependent. These data identify a new mechanism for heparanase in promoting the release of pro-inflammatory cytokines that is likely to be important in regulating cell migration and inflammation.     

Purification and characterization of heparan sulfate from human primary osteoblasts

Heparan sulfate (HS) is a linear, highly variable, highly sulfated glycosaminoglycan sugar whose biological activity largely depends on internal sulfated domains that mediate specific binding to an extensive range of proteins. In this study we employed anion exchange chromatography, molecular sieving and enzymatic cleavage on HS fractions purified from three compartments of cultured osteoblasts—soluble conditioned media, cell surface, and extracellular matrix (ECM). We demonstrate that the composition of HS chains purified from the different compartments is structurally non‐identical by a number of parameters, and that these differences have significant ramifications for their ligand‐binding properties. The HS chains purified of conditioned medium had twice the binding affinity for FGF2 when compared with either cell surface or ECM HS. In contrast, similar binding of BMP2 to the three types of HS was observed. These results suggest that different biological compartments of cultured cells have structurally and functionally distinct HS species that help to modulate the flow of HS‐dependent factors between the ECM and the cell surface. J. Cell. Biochem. 108: 1132–1142, 2009. © 2009 Wiley‐Liss, Inc.     

Release of cell fragments by invading melanoma cells

Tumor cell invasion requires coordinated cell adhesion to an extracellular matrix (ECM) substrate at the leading edge and concomitant detachment at the cell rear. Known detachment mechanisms include the slow sliding of focal contacts, the detachment of adhesion receptors by affinity and avidity regulation, as well as the shedding of adhesion receptors, most notably integrins. In highly invasive melanoma cells migrating within 3D collagen matrices, beta1 integrins and CD44 are released upon retraction of the trailing edge, together with ripping-off complete cell fragments to become deposited along the migration trail of remodeled matrix. Cell fragments reach a size up to 12 microm in diameter, contain cytoplasm and occasionally polymerized actin enclosed by intact cell membrane including surface beta1 integrins, but do not include nuclear material. The release of cell fragments was migration dependent, as impairment of motility by a blocking anti-beta1 integrin antibody also blocked cell particle release. Invasion-associated deposition of cell fragments combines the secretory-type release of vesicles with a physical mechanism of rear retraction and migration efficiency. The deposition of cell fragments may further represent a disregulated detachment strategy with implications for neoplastic cell behavior, such as the paracrine effects on neighbor cells or a negative impact on immune effector cells.     

The good and the bad of chemokines/chemokine receptors in melanoma

Chemokine ligand/receptor interactions affect melanoma cell growth, stimulate or inhibit angiogenesis, recruit leukocytes, promote metastasis, and alter the gene expression profile of the melanoma associated fibroblasts. Chemokine/chemokine receptor interactions can protect against tumor development/growth or can stimulate melanoma tumor progression, tumor growth and metastasis. Metastatic melanoma cells express chemokine receptors that play a major role in the specifying the organ site for metastasis, based upon receptor detection of the chemokine gradient elaborated by a specific organ/tissue. A therapeutic approach that utilizes the protective benefit of chemokines involves delivery of angiostatic chemokines or chemokines that stimulate the infiltration of cytotoxic T cells and natural killer T cells into the tumor microenvironment. An alternative approach that tackles the tumorigenic property of chemokines uses chemokine antibodies or chemokine receptor antagonists to target the growth and metastatic properties of these interactions. Based upon our current understanding of the role of chemokine‐mediated inflammation in cancer, it is important that we learn to appropriately regulate the chemokine contribution to the tumorigenic ‘cytokine/chemokine storm’, and to metastasis.     

Specific degradation of subendothelial matrix proteoglycans by brain-metastatic melanoma and brain endothelial cell heparanases

One of the many features of the malignant phenotype, in vitro and in vivo, is elevated heparanase production and activity. Using in vitro model systems, we examined the capacity of murine (B16B15b) and human (70W) brain-metastatic melanoma cells to degrade the subendothelial matrix produced by endothelial cell monolayer cultures. B16B15b and 70W melanoma cells solubilized sulfated matrix proteoglycans at levels significantly higher than their parental lines (B16F1, MeWo). Sulfated matrix proteoglycans were rich in heparan sulfate (HSPGs), with minor amounts of chondroitin and dermatan sulfates. When matrix HSPGs were treated with pronase and alkaline borohydride to cleave the core proteins, the resulting glycosaminoglycan chains (GAGs) had an estimated M_r of ∼2.7 × 10⁴ Da, with a minor subpopulation possessing an M_r of ∼4.5 × 10⁴ Da. After their incubation with brain-metastatic melanoma cells, new HS fragments with lower M_r estimated at ∼9 × 10³ Da were detected. This confirms action in these cells of heparanase, which is capable of cleaving GAGs at specific intrachain sites and releasing fragments of a relatively high M_r. The pattern of HSPG degradation by brain-metastatic melanoma cells differed from that of less metastatic parental cells or cells metastatic to organs other than the brain. Moreover, supraadditive levels of heparanase activity were found when brain endothelial cells were coincubated with brain-metastatic melanoma cells in equicellular amounts. Cooperative interactions between heparanases from tumor and endothelial sources in the invasion process are suggested and their potential mechanisms discussed. J. Cell. Physiol. 172:334–342, 1997. © 1997 Wiley-Liss, Inc.     

HIP/RPL29 antagonizes VEGF and FGF2 stimulated angiogenesis by interfering with HS-dependent responses

HIP/RPL29 is a heparan sulfate (HS) binding protein with diverse activities including modulation of heparanase (HPSE) activity. We examined HIP/RPL29's ability to modulate actions of HS-binding growth factors (HBGFs) in angiogenesis. Between 1 and 2.5 microg/ml (ca. 60-150 nM), HIP/RPL29 inhibited HBGF-stimulated endothelial cell tube formation. Aortic explant outgrowth also was inhibited, but at higher concentrations (40 microg/ml). At this concentration, HIP/RPL29 had no effect on HBGF-stimulated MAPK phosphorylation or VEGF-stimulated receptor-2 phosphorylation at site Y-996. Partial inhibition occurred at VEGF receptor-2 site Y951, associated with cell migration. HBGF displacement from HS-bearing perlecan domain I showed that HIP/RPL29 released 50% of bound HBGF at 20 microg/ml, a dose where endothelial tube formation is inhibited. Similar FGF2 release occurred at pH 5.0 and 7.0, conditions where HPSE is highly and residually active, respectively. We considered that HIP/RPL29 inhibits HPSE-dependent release of HS-bound HBGFs. At pH 5.0, release of soluble HS was inhibited by 64% at concentrations of 5 microg/ml and by 77% at 40 microg/ml, indicating that HIP/RPL29 antagonizes HPSE activity. At concentrations up to 40 microg/ml (ca. 2.5 microM) where angiogenic processes are inhibited, release of FGF2 occurred in the presence of HPSE and HIP/RPL29. The majority of this FGF2 is not bound to soluble HS. Studies of HIP/RPL29 binding to HS indicated that many structural features of HS are important in modulation of HBGF activities. Our findings suggest that inhibition of angiogenic processes by HIP/RPL29 involves attenuation of the formation of soluble, biologically active HBGF:HS complexes that activate HBGF receptors.     

Virally-induced upregulation of heparan sulfate on B cells via the action of type I IFN

Cell surface heparan sulfate (HS) is an important coreceptor for many cytokines, chemokines, and growth factors. In this study, we report that splenic murine B cells express very little HS and that upon infection with either gammaherpesvirus (murine gammaherpesvirus 68) or betaherpesvirus (murine cytomegalovirus), HS is rapidly upregulated at the surface of B cells. HS upregulation was not observed in mice deficient for the type I IFN (IFN-I) receptor. Additionally, treatment of wild-type mice with the IFN-I inducer polyinosine polycytidylic acid triggered HS expression at the B cell surface. Similarly, incubation of purified splenic B cells with IFN-I, TLR ligands, or BCR stimulators ex vivo resulted in a drastic increase in HS surface expression. We found that IFN-I induced an increase in the surface expression of HS-modified syndecan 4 as well as that of an unidentified heparan sulfate proteoglycan. Finally, IFN-I treatment increased B cell responsiveness to APRIL, a cytokine involved in B cell survival and T cell-independent B cell responses. Enzymatic removal of HS from IFN-I-treated B cells inhibited APRIL. Altogether, our results indicate that upon herpesvirus infection in mice, HS is rapidly upregulated at the surface of B cells due to the action of IFN-I, potentially increasing B cell responsiveness to cytokines. Induction of HS expression at the B cell surface by stimulators of the innate immune response likely plays a key role in the development of a robust immune response.     

Decorin and chondroitin-6 sulfate inhibit B16V melanoma cell migration and invasion by cellular acidification

In vivo, cells are embedded in an environment generated and maintained by multiple cell-cell and cell-matrix interactions. While transiting the dermis metastasizing melanoma cells interact with the extracellular matrix (ECM) and fibroblasts. To study the roles of ECM components and fibroblasts in melanoma (B16V) cell migration and invasion, we established a co-culture system consisting of fibroblasts, their collagen-rich matrix and B16V cells. The crosstalk between B16V cells and fibroblasts was indicated by a clear increase in release and activity of matrix-metallo-protease-2. Time-lapse microscopy revealed that in B16V cells exposed to either decorin or chondroitin sulfates migration and invasion decreased by more than 50%. Decorin led to a reversible, chondroitin-6-sulfate to an irreversible, cytosolic acidification of B16V cells. Interestingly, decorin lowered NHE1 activity whereas chondroitin-6-sulfate did not. Furthermore, decorin and chondroitin-6-sulfate also acidified the pH at the cell surface which might prevent migration due to strong adhesion. In conclusion, the present co-culture system is an appropriate tool to analyze migration, invasion, and MMP release depending on cell-matrix interactions and the crosstalk between the invasive cells and those surrounded by their self-made matrix. We show a so far unknown function of decorin and chondroitin-6-sulfate: their ability to inhibit B16V cell migration by intracellular acidification.     

Novel anti-angiogenic peptides derived from ELR-containing CXC chemokines

Angiogenesis is tightly regulated by numerous endogenous pro- and anti-angiogenic proteins and peptides. Among these are the CXC chemokines, a set of multifunctional peptides. CXC chemokines containing the ELR motif act as pro-angiogenic agents by regulating both endothelial cell proliferation and migration. Here we show that a set of six 22-24-amino acid peptides derived from the pro-angiogenic ELR-containing CXC chemokines exhibit notable anti-proliferative and anti-migratory activity in vitro; we call these peptides chemokinostatins. The ability of the identified peptides to inhibit the basic components of angiogenesis even though they are derived from pro-angiogenic proteins contributes towards the understanding of the diverse role of the CXC chemokine family in angiogenesis.     

Imatinib targets PDGF signaling in melanoma and host smooth muscle neighboring cells

In previous in vitro studies, we showed that imatinib abrogated platelet‐derived growth factor receptor α (PDGFRα) signaling, disrupting both breast cancer and smooth muscle cells (SMC). PDGF is also a powerful mitogen for neural crest origin cells like melanocytes. The purpose of the present study was to evaluate the effect of imatinib on melanoma growth and in angiogenesis, with emphasis to the involvement in PDGF signaling. B16 melanoma cells incubation with 5 µM (IC50) imatinib resulted in a significant reduction in cell proliferation and migration. Apoptosis, however, was not significantly affected. Phosphorylated‐PDGFRα expression was decreased in B16 lysates. In a mouse model of B16 melanoma, intraperitoneal administration of imatinib at early day light significantly decreased tumor growth. These findings were corroborated by a highly significant reduction in cell proliferation and increase in apoptosis in melanoma tumors. This was accompanied by a decrease in microvessel density and in the number of SMC‐presenting vessels. Imatinib further inhibited PDGFRα expression and activity, as confirmed by the down‐regulation of downstream Erk signaling pathway. Altogether, this study demonstrates that besides targeting tumor cells, imatinib also prevents vascular integrity. The current study provides evidence that the paracrine crosstalk between tumor cells and host neighboring cells is crucial for the elucidation of imatinib effects. In addition, the fact that this molecule targets vascular support cells further enlarges its therapeutic purpose to a wide range of vasculoproliferative pathologies. J. Cell. Biochem. 111: 433–441, 2010. © 2010 Wiley‐Liss, Inc.     

Induction of angiogenesis by a fragment of human tyrosyl-tRNA synthetase

The first step of protein synthesis is catalyzed by aminoacyl-tRNA synthetases. In addition, certain mammalian tRNA synthetases link protein synthesis to cytokine signaling pathways. In particular, human tyrosyl-tRNA synthetase (TyrRS) can be split by proteolysis into two fragments having distinct cytokine activities. One of the TyrRS fragments (mini TyrRS) contains features identical to those in CXC chemokines (like interleukin-8) that also act as angiogenic factors. Here mini TyrRS (but not full-length TyrRS) is shown to stimulate chemotaxis of endothelial cells in vitro and stimulate angiogenesis in each of two in vivo animal models. The angiogenic activity of mini TyrRS can be opposed by anti-angiogenic chemokines like IP-10. Thus, a biological fragment of human tyrosyl-tRNA synthetase links protein synthesis to regulation of angiogenesis.     

Granzyme B cleavage of fibronectin disrupts endothelial cell adhesion,migration and capillary tube formation

Dysregulated angiogenesis contributes to the pathogenesis of chronic inflammatory diseases. Modulation of the extracellular matrix by immune-derived proteases can alter endothelial cell–matrix interactions as well as endothelial cell sprouting, migration and capillary formation. Granzyme B is a serine protease that is expressed by a variety of immune cells, and accumulates in the extracellular milieu in many chronic inflammatory disorders that are associated with dysregulated angiogenesis. Although granzyme B is known to cleave fibronectin, an essential glycoprotein in vascular morphogenesis, the role of granzyme B in modulating angiogenesis is unknown. In the present study, granzyme B cleaved both plasma fibronectin and cell-derived fibronectin, resulting in the release of multiple fibronectin fragments. Granzyme B cleavage of fibronectin resulted in a dose-dependent reduction in endothelial cell adhesion to fibronectin as well as reduced endothelial cell migration and tubular formation. These events were prevented when granzyme B activity was inhibited by a small molecule inhibitor. In summary, granzyme B-mediated cleavage of fibronectin contributes to attenuated angiogenesis through the disruption of endothelial cell — fibronectin interaction resulting in impaired endothelial cell migration and tubular formation.     

Fragments generated upon extracellular matrix remodeling: Biological regulators and potential drugs

The remodeling of the extracellular matrix (ECM) by several protease families releases a number of bioactive fragments, which regulate numerous biological processes such as autophagy, angiogenesis, adipogenesis, fibrosis, tumor growth, metastasis and wound healing. We review here the proteases which generate bioactive ECM fragments, their ECM substrates, the major bioactive ECM fragments, together with their biological properties and their receptors. The translation of ECM fragments into drugs is challenging and would take advantage of an integrative approach to optimize the design of pre-clinical and clinical studies. This could be done by building the contextualized interaction network of the ECM fragment repertoire including their parent proteins, remodeling proteinases, and their receptors, and by using mathematical disease models.     

Identification and quantification of proteins differentially secreted by a pair of normal and malignant breast‐cancer cell lines

Secreted proteins play a pivotal role in cellular functions. To better understand malignant behavior, we adapted stable isotopic labeling with amino acids in cell culture technology to identify and quantify proteins differentially released into the extracellular media by a pair of normal and malignant breast‐cancer cell lines. Approximately 380 non‐redundant proteins were quantified in serum‐free media. Of the assigned proteins, 62% are classified secreted in protein databases and an additional 25% are designated secreted in the literature. A number of growth factors were found differentially regulated. Tumor necrosis factor, pigment epithelial‐differentiating factor and stem‐cell growth factor precursor showed decreased expression in breast‐cancer cell line, whereas Inhibin beta and macrophage migration inhibitory factor show increased expression. Interestingly, protease inhibitors, including plasma protease (C1) inhibitor, PZP precursor, and SerpinE2 were significantly down‐regulated in cancer cell line as were angiostatic factors from extracellular matrix (ECM) such as endorepillin. Further, the C‐terminal fragment of type XVIII collagen, endostatin, a potent angiostatic factor, was down‐regulated as well whereas extracellular collagens and osteoblast‐specific factor 2 (OSF‐2), were up‐regulated. Differential expression and secretion of SerpinE2 and OSF‐2 were confirmed using Western blotting. These results corroborate models of invasive tumors sustained by elaborate coordination of stromal cells via chemokines and growth factors, while protease inhibitors remodel the ECM to stimulate angiogenesis.     

Study of motogenic signal in human melanoma cells

The components of the extracellular matrix (ECM) are more than just adhesion sites for migrating tumor cells: following enzymatic degradation of the ECM, the release of sequestrated growth factors increases, thus they become available for tumor cells. In a number of cancers dysfunction of epidermal growth factor receptor (EGFR) or hepatocyte growth factor receptor (c-Met) contribute to the malignant transformation that directly regulates cell proliferation, survival and motility. Furthermore, intracellular calcium level plays an important role in the regulation of the tyrosine kinase pathway. In our preclinical experiments, by administering heparin-derived oligosaccharides we influenced the interaction between human melanoma cells and ECM. In vitro cell migration was inhibited by heparin fragments. Moreover, two of the effective oligosaccharides reduced the number of lung colonies formed in SCID mice. In human melanoma cells an important element of Ca2+ homeostasis, the purinergic Ca2+ channel P2X7 proved to be an anti-apoptotic protein. EGFR and c-Met showed constitutive activity in human melanoma cells, and their inhibition in vitro caused decreased proliferation, migration and elevated apoptosis. Administration of a selective c-Met-TKI significantly decreased primary tumor growth in vivo as well as the capacity for liver colony formation in SCID mice. Selective EGFR-TKI had less inhibitory effect on metastasis formation, and had no effect on the primary tumor. Our results suggest the necessity of a rational dual-specific drug design for the purpose in the therapy of malignant melanoma.     

Heparanase expression and function during early pregnancy in mice

Embryo implantation is a complex process that involves interactions between cell-surface and extracellular components of the blastocyst and the uterus, including blastocyst adhesion to the uterine luminal epithelium, epithelial basement membrane penetration and stromal extracellular matrix remodeling, angiogenesis, and decidualization. These processes all involve interactions with heparan sulfate (HS) proteoglycans, which harbor various growth factors and cytokines and support cell adhesion. Heparanase (HPSE) is an endo-beta-glucuronidase that cleaves HS at specific sites. HPSE also can act as an adhesion molecule independent of its catalytic activity. Thus, HPSE is a multifunctional molecule contributing to and modulating HS-dependent processes. Exogenously added HPSE improves embryo implantation in mice; however, no information is available regarding the normal pattern of HPSE expression and activity during the implantation process in any system. Using several approaches, including real-time RT-PCR, in situ hybridization, and immunohistochemistry, we determined that uterine HPSE expression increases dramatically during early pregnancy in mice. Heparanase mRNA and protein were primarily expressed in decidua and were rapidly induced at the implantation site. Uterine HPSE activity was characterized and demonstrated to increase >40-fold during early pregnancy. Finally, we demonstrate that the HPSE inhibitor PI-88 severely inhibits embryo implantation in vivo. Collectively, these results indicate that HPSE plays a role in blastocyst implantation and complements previous studies showing a role for HS-dependent interactions in this process.