Ralph D. Sanderson

HEPARAN SULFATE PROTEOGLYCANS AS ADHESIVE AND ANTI-INVASIVE MOLECULES : SYNDECANS AND GLYPICAN HAVE DISTINCT FUNCTIONS

ARH-77 cells do not adhere to type I collagen and readily invade into collagen gels, but following expression of the transmembrane heparan sulfate proteoglycan syndecan-1, they bind collagen and fail to invade. We now show that cells transfected with syndecan-2 or syndecan-4 also bind collagen and are non-invasive. In contrast, cells transfected with the glycosylphosphatidylinositol-anchored proteoglycan glypican-1 do not bind to collagen and remain invasive, even though glypican- and syndecan-expressing cells have similar surface levels of heparan sulfate, and their proteoglycans have similar affinities for collagen. Analysis of cells expressing syndecan-1-glypican-1 chimeric proteoglycans reveals that inhibition of invasion requires the extracellular domain of syndecan but not its transmembrane or cytoplasmic domain. Surprisingly, cells bearing a chimera composed of the glypican extracellular domain fused to the syndecan transmembrane and cytoplasmic domains bind to collagen but remain invasive, implying that adhesion to collagen is not by itself sufficient to inhibit invasion. Apparently, the extracellular domain of syndecan-1, presumably by interacting with cell-surface signal transducing molecules, directly regulates complex cell behaviors such as motility and invasiveness. These results also show for the first time that syndecans and glypicans can have distinct functions, even when expressed by the same cell type.

Heparanase stimulation of protease expression implicates it as a master regulator of the aggressive tumor phenotype in myeloma.

High levels of heparanase are an indicator of poor prognosis in myeloma patients, and up-regulation of the enzyme enhances tumor growth, angiogenesis, and metastasis in animal models. At least part of the impact of heparanase in driving the aggressive tumor phenotype is due to its effect on increasing the expression and shedding of the heparan sulfate proteoglycan syndecan-1, a molecule known to promote myeloma progression. The present work demonstrated that elevation in heparanase expression in myeloma cells stimulates sustained ERK phosphorylation that in turn drives MMP-9 expression. In addition, urokinase-type plasminogen activator (uPA) and uPA receptor expression levels increased, and blocking the proteolytic activation of either MMP-9 or uPA inhibited the heparanase-induced increase in syndecan-1 shedding. Together these data provide a mechanism for heparanase-induced syndecan-1 shedding and, more importantly, demonstrate that heparanase activity in myeloma cells can lead to increased levels of proteases that are known to play important roles in the aggressive behavior of myeloma tumors. This in addition to its other known biological roles, indicates that heparanase acts as a master regulator of the aggressive tumor phenotype by up-regulating protease expression and activity within the tumor microenvironment.

Enzymatic remodeling of heparan sulfate proteoglycans within the tumor microenvironment: Growth regulation and the prospect of new cancer therapies

Heparan sulfate proteoglycans (HSPGs), via their interactions with numerous effector molecules such as FGF‐2, IL‐8, and VEGF, regulate the biological activity of cells by acting as co‐receptors that promote signaling. The extent and nature of their role as co‐receptors is often misregulated in cancer as manifested by alterations in HSPG structure and expression level. This misregulation of HSPGs can aid in promoting the malignant phenotype. In addition to expression‐related changes in HSPGs, recent discoveries indicate that HSPGs localized within the tumor microenvironment can be attacked by enzymes that alter proteoglycan structure resulting in dramatic effects on tumor growth and metastasis. This review focuses on remodeling of HSPGs by three distinct mechanisms that occur in vivo; (i) shedding of proteoglycan extracellular domains from cell surfaces, (ii) fragmentation of heparan sulfate chains by heparanase, and (iii) removal of sulfates from the 6‐O position of heparan sulfate chains by extracellular sulfatases. Assessing or monitoring the remodeling of HSPGs has important implications for tumor diagnosis and patient prognosis while therapeutic manipulation of the remodeling process represents an exciting new possibility for treating cancer. J. Cell. Biochem.

HSulf-1 and HSulf-2 Are Potent Inhibitors of Myeloma Tumor Growth in Vivo

To participate as co-receptor in growth factor signaling, heparan sulfate must have specific structural features. Recent studies show that when the levels of 6-O-sulfation of heparan sulfate are diminished by the activity of extracellular heparan sulfate 6-O-endosulfatases (Sulfs), fibroblast growth factor 2-, heparin binding epidermal growth factor-, and hepatocyte growth factor-mediated signaling are attenuated. This represents a novel mechanism for regulating cell growth, particularly within the tumor microenvironment where the Sulfs are known to be misregulated. To directly test the role of Sulfs in tumor growth control in vivo, a human myeloma cell line was transfected with cDNAs encoding either of the two known human endosulfatases, HSulf-1 or HSulf-2. When implanted into severe combined immunodeficient (SCID) mice, the growth of these tumors was dramatically reduced on the order of 5- to 10-fold as compared with controls. In addition to an inhibition of tumor growth, these studies revealed the following. (i) HSulf-1 and HSulf-2 have similar functions in vivo. (ii) The extracellular activity of Sulfs is restricted to the local tumor cell surface. (iii) The Sulfs promote a marked increase in extracellular matrix deposition within tumors that may, along with attenuated growth factor signaling, contribute to the reduction in tumor growth. These findings demonstrate that dynamic regulation of heparan sulfate structure by Sulfs present within the tumor microenvironment can have a dramatic impact on the growth and progression of malignant cells in vivo.

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-β-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.

Syndecan-1 (CD138) Immunoreactivity in Bone Marrow Biopsies of Multiple Myeloma: Shed Syndecan-1 Accumulates in Fibrotic Regions

Syndecan-1 (CD138) mediates myeloma cell adhesion, and loss of syndecan-1 from the cell surface may contribute to myeloma proliferation and dissemination. Flow cytometry analysis of myeloma cells in bone marrow specimens shows heterogeneity in cell surface syndecan-1 expression. It is not known whether weaker expression correlates with more aggressive disease. However, recent reports suggest that variations in syndecan-1 staining intensity on myeloma cells may be an artifact of specimen handling. In this study, we evaluate syndecan-1 expression in bone marrow biopsy sections from 28 multiple myeloma patients, to elucidate the heterogeneity of syndecan-1 expression in situ. Immunoreactivity for syndecan-1, using the antibody B-B4 (CD138), was found in more than 95% of multiple myeloma cells in 27 of 28 biopsies. However, one biopsy had more than 50% CD138-negative cells and cells with weak CD138 expression were identified in the majority of cases. Loss of syndecan-1 did not appear to relate to myeloma cell differentiation. In addition, syndecan-1 was detected on intravascular and intrasinusoidal myeloma cells suggesting that loss of syndecan-1 may not be required for extramedullary dissemination. Bone marrow biopsies from nine additional patients, with variable CD138 staining intensity on myeloma cells as determined by flow cytometry, were studied by immunohistochemistry. The heterogenous CD138 expression was confirmed in situ, with weakly positive cells concentrated in areas of reticulin fibrosis. These cells had a disrupted pattern of membrane staining in contrast to the strong linear membrane staining seen in the other multiple myeloma cells. In addition, the fibrotic stroma stained intensely for syndecan-1. Accumulation of syndecan-1 within the extracellular matrix of the marrow likely is derived by shedding of the molecule from the surface of myeloma cells. Because syndecan-1 can act to regulate the activity of heparan-binding growth factors, these reservoirs of syndecan-1 may play a critical role in promoting myeloma pathogenesis, or in regeneration of the tumor after chemotherapy.

Multiple Heparan Sulfate Chains Are Required for Optimal Syndecan-1 Function

Syndecans have three highly conserved sites available for heparan sulfate attachment. To determine if all three sites are required for normal function, a series of mutated syndecans having two, one, or no heparan sulfate chains were expressed in ARH-77 cells. Previously, we demonstrated that expression of wild-type syndecan-1 on these myeloma cells mediates cell-matrix and cell-cell adhesion and inhibits cell invasion into collagen gels. Here we show that to optimally mediate each of these activities, all three sites of heparan sulfate attachment are required. Generally, an increasing loss of syndecan-1 function occurs as the number of heparan sulfate attachment sites decreases. This loss of function is not the result of a decrease in either the total amount of cell surface heparan sulfate or syndecan-1 core protein. In regard to cell invasion, cells expressing syndecan-1 bearing a single heparan sulfate attachment site exhibit a hierarchy of function based upon the position of the site within the core protein; the presence of an available attachment site at serine 47 confers the greatest level of activity, while serine 37 contributes little to syndecan-1 function. However, when all three heparan sulfate chains are present, significantly greater biological activity is observed than is predicted by the sum of the activities occurring when the chains act individually. This synergy provides a functional basis for the evolutionary conservation of the three heparan sulfate attachment sites on syndecans and supports the idea that molecular heterogeneity, which is characteristic of proteoglycans, contributes to their functional diversity.

Elevated levels of shed syndecan-1 correlate with tumour mass and decreased matrix metalloproteinase-9 activity in the serum of patients with multiple myeloma

Sera from 20 myeloma patients and 12 normal controls were analysed for the presence of syndecan‐1 and matrix metalloproteinase‐9 (MMP‐9). The level of syndecan‐1 in the serum was elevated in 7/20 (35%) myeloma patients whilst 6/19 patients (31%) had decreased serum MMP‐9 activity. The presence of increased syndecan‐1 was associated with decreased serum MMP‐9. Both elevated syndecan‐1 and decreased MMP‐9 were associated with higher marrow plasmacytosis, serum beta‐2 microglobulin and paraprotein levels. These data provide evidence that the syndecan‐1 ectodomain is shed in vivo. Quantitation of serum syndecan‐1 may be a useful measure of tumour mass and may have important implications for myeloma biology.

Syndecan-1 Is Required for Robust Growth, Vascularization, and Metastasis of Myeloma Tumors in Vivo

Myeloma tumors are characterized by high expression of syndecan-1 (CD138), a heparan sulfate proteoglycan present on the myeloma cell surface and shed into the tumor microenvironment. High levels of shed syndecan-1 in the serum of patients are an indicator of poor prognosis, and numerous studies have implicated syndecan-1 in promoting the growth and progression of this cancer. In the present study we directly addressed the role of syndecan-1 in myeloma by stable knockdown of its expression using RNA interference. Knockdown cells that were negative for syndecan-1 expression became apoptotic and failed to grow in vitro. Knockdown cells expressing syndecan-1 at ∼28% or ∼14% of normal levels survived and grew well in vitro but formed fewer and much smaller subcutaneous tumors in mice compared with tumors formed by cells expressing normal levels of syndecan-1. When injected intravenously into mice (experimental metastasis model), knockdown cells formed very few metastases as compared with controls. This indicates that syndecan-1 may be required for the establishment of multi-focal metastasis, a hallmark of this cancer. One mechanism of syndecan-1 action occurs via stimulation of tumor angiogenesis because tumors formed by knockdown cells exhibited diminished levels of vascular endothelial growth factor and impaired development of blood vessels. Together, these data indicate that the effects of syndecan-1 on myeloma survival, growth, and dissemination are due, at least in part, to its positive regulation of tumor-host interactions that generate an environment capable of sustaining robust tumor growth.

Heparan Sulfate Chains of Syndecan-1 Regulate Ectodomain Shedding

Background: Syndecan ectodomains shed by cells can enhance progression of cancer, inflammatory disease, and pathogen infection. Results: Reducing the amount of heparan sulfate present on syndecan core proteins increases shedding of the ectodomain. Conclusion: Heparan sulfate chains suppress syndecan shedding. Significance: Therapeutic inhibition of heparan sulfate degradation could slow disease progression. Matrix metalloproteinases release intact syndecan-1 ectodomains from the cell surface giving rise to a soluble, shed form of the proteoglycan. Although it is known that shed syndecan-1 controls diverse pathophysiological responses in cancer, wound healing, inflammation, infection, and immunity, the mechanisms regulating shedding remain unclear. We have discovered that the heparan sulfate chains present on syndecan core proteins suppress shedding of the proteoglycan. Syndecan shedding is dramatically enhanced when the heparan sulfate chains are enzymatically degraded or absent from the core protein. Exogenous heparan sulfate or heparin does not inhibit shedding, indicating that heparan sulfate must be attached to the core protein to suppress shedding. Regulation of shedding by heparan sulfate occurs in multiple cell types, for both syndecan-1 and syndecan-4 and in murine and human syndecans. Mechanistically, the loss of heparan sulfate enhances the susceptibility of the core protein to proteolytic cleavage by matrix metalloproteinases. Enhanced shedding of syndecan-1 following loss of heparan sulfate is accompanied by a dramatic increase in core protein synthesis. This suggests that in response to an increase in the rate of shedding, cells attempt to maintain a significant level of syndecan-1 on the cell surface. Together these data indicate that the amount of heparan sulfate present on syndecan core proteins regulates both the rate of syndecan shedding and core protein synthesis. These findings assign new functions to heparan sulfate chains, thereby broadening our understanding of their physiological importance and implying that therapeutic inhibition of heparan sulfate degradation could impact the progression of some diseases.