heparitin-sulfate and Multiple-Myeloma

Plasma cells no longer express a B-cell antigen receptor and are hence deprived of signals crucial for survival throughout B-cell development. Instead, normal plasma cells, as well as their malignant myeloma counterparts, heavily rely on communication with the bone marrow (BM) microenvironment for survival. The plasma cell heparan sulfate proteoglycan (HSPG) syndecan-1 (CD138) and HSPGs in the BM microenvironment act as master regulators of this communication by co-opting specific growth and survival factors from the BM niche. This designates syndecan-1/HSPGs and their synthesis machinery as potential treatment targets in multiple myeloma.

Topics: Animals; Bone Marrow; Heparitin Sulfate; Humans; Multiple Myeloma; Plasma Cells; Proteoglycans; Syndecan-1; Tumor Microenvironment

Heparanase is an endo-β-D-glucuronidase capable of cleaving heparan sulfate side chains at a limited number of sites, yielding heparan sulfate fragments of still appreciable size. Importantly, heparanase activity correlates with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of heparan sulfate cleavage and remodeling of the extracellular matrix and basement membrane underlying epithelial and endothelial cells. Similarly, heparanase activity is implicated in neovascularization, inflammation and autoimmunity, involving the migration of vascular endothelial cells and activated cells of the immune system. The cloning of a single human heparanase cDNA 10 years ago enabled researchers to critically approve the notion that heparan sulfate cleavage by heparanase is required for structural remodeling of the extracellular matrix, thereby facilitating cell invasion. Progress in the field has expanded the scope of heparanase function and its significance in tumor progression and other pathologies. Notably, although heparanase inhibitors attenuated tumor progression and metastasis in several experimental systems, other studies revealed that heparanase also functions in an enzymatic activity-independent manner. Thus, inactive heparanase was noted to facilitate adhesion and migration of primary endothelial cells and to promote phosphorylation of signaling molecules such as Akt and Src, facilitating gene transcription (i.e. vascular endothelial growth factor) and phosphorylation of selected Src substrates (i.e. endothelial growth factor receptor). The concept of enzymatic activity-independent function of heparanase gained substantial support by the recent identification of the heparanase C-terminus domain as the molecular determinant behind its signaling capacity. Identification and characterization of a human heparanase splice variant (T5) devoid of enzymatic activity and endowed with protumorigenic characteristics, elucidation of cross-talk between heparanase and other extracellular matrix-degrading enzymes, and identification of single nucleotide polymorphism associated with heparanase expression and increased risk of graft versus host disease add other layers of complexity to heparanase function in health and disease.

Topics: Antineoplastic Agents; Disease Progression; Enzyme Inhibitors; ErbB Receptors; Glucuronidase; Head and Neck Neoplasms; Heparitin Sulfate; Humans; Multiple Myeloma; Neoplasm Metastasis; Neoplasms; Proteoglycans; Signal Transduction

Heparanase activity is strongly implicated in structural remodeling of the extracellular matrix, a process which can lead to invasion by tumor cells. In addition, heparanase augments signaling cascades leading to enhanced phosphorylation of selected protein kinases and increased gene transcription associated with aggressive tumor progression. This function is apparently independent of heparan sulfate and enzyme activity, and is mediated by a novel protein domain localized at the heparanase C-terminus. Moreover, the functional repertoire of heparanase is expanded by its regulation of syndecan clustering, shedding, and mitogen binding. Recent reports indicate that modified glycol-split heparin, which inhibits heparanase activity, can profoundly inhibit the progression of tumor xenografts produced by myeloma and carcinoma cells, thus moving anti-heparanase therapy closer to reality.

Topics: Animals; Cell Adhesion; Endocytosis; Enzyme Activation; Extracellular Matrix; Glucuronidase; Heparin; Heparitin Sulfate; Humans; Multiple Myeloma; Neoplasms; rac GTP-Binding Proteins; Receptors, Cell Surface; Signal Transduction; src-Family Kinases; Structure-Activity Relationship; Substrate Specificity; Syndecan-1

Emerging data in myeloma and other cancers indicates that heparan sulfate proteoglycans promote tumor progression by enhancing their growth and metastasis. By acting as key regulators of cell signaling via their interactions with multiple growth and angiogenic factors, heparan sulfates mediate a shift in the microenvironment that supports the tumor as an 'organ' and promotes an aggressive tumor phenotype. In addition, enzymatic remodeling of heparan sulfate proteoglycans provides a mechanism for rapid, localized and dynamic modulation of proteoglycan function thereby tightly regulating activities within the tumor microenvironment. New data from animal models demonstrates that heparan sulfate or the enzymes that regulate heparan sulfate are viable targets for cancer therapy. This strategy of targeting heparan sulfate may be particularly effective for attacking cancers like myeloma where extensive genetic chaos renders them unlikely to respond well to agents that target a single signaling pathway.

Topics: Animals; Cell Proliferation; Glucuronidase; Heparitin Sulfate; Humans; Mice; Multiple Myeloma; Neoplasm Metastasis; Signal Transduction; Sulfatases; Syndecan-1

This review summarizes a series of studies demonstrating that heparan sulfate proteoglycans act to promote the growth and metastasis of myeloma and breast tumors, two tumors that home to, and grow within, bone. Much of the growth-promoting effect of proteoglycans in these tumors may reside in the shed form of syndecan-1 that acts to favorably condition the tumor microenvironment. Moreover, the interplay between heparan sulfate and the extracellular enzyme heparanase-1 also has important regulatory implications. Recent studies indicate that the activity of heparanase, which likely releases heparin sulfate-bound growth factors and generates highly active heparan sulfate fragments, also promotes growth and metastasis of myeloma and breast tumors. Understanding the role of heparan sulfate and heparanase in the regulation of tumor behavior may lead to new therapeutic approaches for treating cancer.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Cell Proliferation; Glucuronidase; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Models, Biological; Multiple Myeloma; Neoplasm Metastasis; Osteolysis

Topics: CD57 Antigens; Exostoses; Genes, Tumor Suppressor; Heparitin Sulfate; Humans; Membrane Proteins; Multiple Myeloma; N-Acetylgalactosaminyltransferases; N-Acetylglucosaminyltransferases; N-Acetylhexosaminyltransferases; Proteoglycans; Recombinant Proteins; Substrate Specificity

Multiple myeloma is a hematological neoplasm derived from plasma cells invariably developing in the bone marrow (BM). The persisting clinical challenge in MM resides in its high ability to resist drugs as shown by the frequent relapses observed in patients regardless of the treatment applied. In a mouse model of MM, we identified a subpopulation of cells harboring increased resistance to current MM drugs. These cells bound a proliferation inducing ligand (APRIL), a key MM promoting/survival factor. APRIL binding involved the heparan sulfate (HS) chain present on syndecan-1 (SDC-1), and correlated with reactivity to the anti-HS antibody 10e4. 10e4

Topics: Animals; Bone Marrow; Heparitin Sulfate; Mice; Multiple Myeloma; Sulfotransferases; Syndecan-1

The survival and proliferation of multiple myeloma (MM) cells in the bone marrow (BM) critically depend on interaction with stromal cells expressing the chemokine CXCL12. CXCL12 regulates the homing to the BM niche by mediating the transendothelial migration and adhesion/retention of the MM cells. The gamma isoform of CXCL12 (CXCL12γ) has been reported to be highly expressed in mouse BM and to show enhanced biological activity compared to the 'common' CXCL12α isoform, mediated by its unique extended C-terminal domain, which binds heparan sulfate proteoglycans (HSPGs) with an extraordinary high affinity. Here, we investigated the expression of CXCL12γ in human BM and studied its functional role in the interaction of MM cells with BM stromal cells (BMSCs).. We assessed CXCL12γ mRNA and protein expression by human BMSCs using qPCR, flow cytometry, and immunohistochemistry. CRISPR-Cas9 was employed to delete CXCL12γ and the heparan sulfate (HS) co-polymerase EXT1 in BMSCs. To study the functional roles of BMSC-derived CXCL12γ and HSPGs in the interaction of MM cells with BMSCs cells, MM cell lines and primary MM cells were co-cultured with BMSCs.. We observed that CXCL12γ is expressed in situ by reticular stromal cells in both normal and MM BM, as well as by primary BMSC isolates and BMSC lines. Importantly, upon secretion, CXCL12γ, unlike the CXCL12α isoform, was retained on the surface of BMSCs. This membrane retention of CXCL12γ is HSPG mediated, since it was completely annulated by CRISPR-Cas9-mediated deletion of the HS co-polymerase EXT1. CXCL12γ expressed by BMSCs and membrane-retained by HSPGs supported robust adhesion of MM cells to the BMSCs. Specific genetic deletion of either CXCL12γ or EXT1 significantly attenuated the ability of BMSCs to support MM cell adhesion and, in addition, impaired their capacity to protect MM cells from bortezomib-induced cell death.. We show that CXCL12γ is expressed by human BMSCs and upon secretion is retained on their cell surface by HSPGs. The membrane-bound CXCL12γ controls adhesion of MM cells to the stromal niche and mediates drug resistance. These findings designate CXCL12γ and associated HSPGs as partners in mediating MM-niche interaction and as potential therapeutic targets in MM.

Topics: Cell Adhesion; Cell Line; Cell Line, Tumor; Chemokine CXCL12; Coculture Techniques; Drug Resistance, Neoplasm; Heparitin Sulfate; Humans; Mesenchymal Stem Cells; Multiple Myeloma; Proteasome Inhibitors

Multiple myeloma (MM) is characterized by the expansion of malignant plasma cells in the bone marrow (BM). Most MMs display aberrant Wnt/β-catenin signaling, which drives proliferation; however, they lack oncogenic Wnt pathway mutations, suggesting activation by autocrine Wnt ligands and/or paracrine Wnts from the BM microenvironment. Expression of the heparan sulfate (HS) proteoglycan syndecan-1 is a hallmark of MM. Syndecan-1 is a critical player in the complex reciprocal interaction between MM cells and their BM niche, mediating growth factor/cytokine binding and signaling by its HS chains. Here, by means of CRISPR/Cas9-mediated knockout and doxycycline-inducible short hairpin RNA-mediated knockdown of EXT1, a critical enzyme for HS polymerization, we demonstrate that the HS chains decorating syndecan-1 mediate aberrant Wnt pathway activation in MM. HS-deficient MM cells exhibited strongly decreased autocrine Wnt/β-catenin pathway activity and reduced Wnt pathway-dependent proliferation. In addition, we demonstrate that Wnts bind to the HS side chains of syndecan-1 and that this binding contributes to paracrine Wnt pathway activation through the Wnt receptor Frizzled (Fzd). Furthermore, in an HS-dependent fashion, syndecan-1 also binds osteoblast-produced R-spondin, which represses Fzd degradation by activation of LGR4, an R-spondin receptor aberrantly expressed on MM cells. Costimulation with R-spondin and its binding to HS chains decorating syndecan-1 are indispensable for optimal stimulation of Wnt signaling in MM. Taken together, our results identify syndecan-1 as a crucial component of the Wnt signalosome in MM cells, binding Wnts and R-spondins to promote aberrant Wnt/β-catenin signaling and cell growth, and suggest HS and its biosynthetic enzymes as potential targets in the treatment of MM.

Topics: beta Catenin; Cell Line, Tumor; Frizzled Receptors; Heparitin Sulfate; Humans; Multiple Myeloma; Neoplasm Proteins; Receptors, G-Protein-Coupled; Syndecan-1; Thrombospondins; Wnt Proteins; Wnt Signaling Pathway

Heparanase is a mammalian endoglycosidase that cleaves heparan sulfate (HS) polysaccharides and contributes to remodelling of the extracellular matrix and regulation of HS-binding protein bioavailabilities. Heparanase is upregulated in malignant cancers and inflammation, aiding cell migration and the release of signaling molecules. It is established as a highly druggable extracellular target for anticancer therapy, but current compounds have limitations, because of cost, production complexity, or off-target effects. Here, we report the synthesis of a novel, targeted library of single-entity glycomimetic clusters capped with simple sulfated saccharides. Several dendrimer HS glycomimetics display low nM IC

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Biomimetic Materials; Cell Line, Tumor; Dendrimers; Enzyme Inhibitors; Fibroblast Growth Factor 2; Glucuronidase; Glycosides; Heparitin Sulfate; Humans; Inhibitory Concentration 50; Mice; Molecular Structure; Multiple Myeloma; Signal Transduction; Xenograft Model Antitumor Assays

Alginate is a promising polysaccharide for use in biomaterials as it is biologically inert. One way to functionalize alginate is by chemical sulfation to emulate sulfated glycosaminoglycans, which interact with a variety of proteins critical for tissue development and homeostasis. In the present work we studied the impact of chain length and flexibility of sulfated alginates for interactions with FGF-2 and HGF. Both growth factors interact with defined sequences of heparan sulfate (HS) at the cell surface or in the extracellular matrix. Whereas FGF-2 interacts with a pentasaccharide sequence containing a critical 2-O-sulfated iduronic acid, HGF has been suggested to require a highly sulfated HS/heparin octasaccharide. Here, oligosaccharides of alternating mannuronic and guluronic acid (MG) were sulfated and assessed by their relative efficacy at releasing growth factor bound to the surface of myeloma cells. 8-mers of sulfated MG (SMG) alginate showed significant HGF release compared to shorter fragments, while the maximum efficacy was achieved at a chain length average of 14 monosaccharides. FGF-2 release required a higher concentration of the SMG fragments, and the 14-mer was less potent compared to an equally sulfated high-molecular weight SMG. Sulfated mannuronan (SM) was subjected to periodate oxidation to increase chain flexibility. To assess the change in flexibility, the persistence length was estimated by SEC-MALLS analysis and the Bohdanecky approach to the worm-like chain model. A high degree of oxidation of SM resulted in approximately twice as potent HGF release compared to the nonoxidized SM alginate. The release of FGF-2 also increased with the degree of oxidation, but to a lower degree compared to that of HGF. It was found that the SM alginates were more efficient at releasing FGF-2 than the SMG alginates, indicating a greater dependence on monosaccharide identity and charge orientation over chain flexibility and charge density.

Topics: Alginates; Cell Line, Tumor; Fibroblast Growth Factor 2; Glucuronic Acid; Glycosaminoglycans; Heparitin Sulfate; Hepatocyte Growth Factor; Hexuronic Acids; Humans; Multiple Myeloma; Oligosaccharides; Sulfates

Emerging evidence indicates that exosomes play a key role in tumor-host cross-talk and that exosome secretion, composition, and functional capacity are altered as tumors progress to an aggressive phenotype. However, little is known regarding the mechanisms that regulate these changes. Heparanase is an enzyme whose expression is up-regulated as tumors become more aggressive and is associated with enhanced tumor growth, angiogenesis, and metastasis. We have discovered that in human cancer cells (myeloma, lymphoblastoid, and breast cancer), when expression of heparanase is enhanced or when tumor cells are exposed to exogenous heparanase, exosome secretion is dramatically increased. Heparanase enzyme activity is required for robust enhancement of exosome secretion because enzymatically inactive forms of heparanase, even when present in high amounts, do not dramatically increase exosome secretion. Heparanase also impacts exosome protein cargo as reflected by higher levels of syndecan-1, VEGF, and hepatocyte growth factor in exosomes secreted by heparanase-high expressing cells as compared with heparanase-low expressing cells. In functional assays, exosomes from heparanase-high cells stimulated spreading of tumor cells on fibronectin and invasion of endothelial cells through extracellular matrix better than did exosomes secreted by heparanase-low cells. These studies reveal that heparanase helps drive exosome secretion, alters exosome composition, and facilitates production of exosomes that impact both tumor and host cell behavior, thereby promoting tumor progression.

Topics: Cell Line, Tumor; Disease Progression; DNA, Complementary; Exosomes; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glucuronidase; Heparitin Sulfate; Hepatocyte Growth Factor; Humans; Multiple Myeloma; Neoplasms; Neovascularization, Pathologic; Syndecan-1; Vascular Endothelial Growth Factor A

The glycome, i.e. the cellular repertoire of glycan structures, contributes to important functions such as adhesion and intercellular communication. Enzymes regulating cellular glycosylation processes are related to the pathogenesis of cancer including multiple myeloma. Here we analyze the transcriptional differences in the glycome of normal (n = 10) and two cohorts of 332 and 345 malignant plasma-cell samples, association with known multiple myeloma subentities as defined by presence of chromosomal aberrations, potential therapeutic targets, and its prognostic impact. We found i) malignant vs. normal plasma cells to show a characteristic glycome-signature. They can ii) be delineated by a lasso-based predictor from normal plasma cells based on this signature. iii) Cytogenetic aberrations lead to distinct glycan-gene expression patterns for t(11;14), t(4;14), hyperdiploidy, 1q21-gain and deletion of 13q14. iv) A 38-gene glycome-signature significantly delineates patients with adverse survival in two independent cohorts of 545 patients treated with high-dose melphalan and autologous stem cell transplantation. v) As single gene, expression of the phosphatidyl-inositol-glycan protein M as part of the targetable glycosyl-phosphatidyl-inositol-anchor-biosynthesis pathway is associated with adverse survival. The prognostically relevant glycome deviation in malignant cells invites novel strategies of therapy for multiple myeloma.

Topics: Cluster Analysis; Gene Dosage; Gene Expression Profiling; Gene Expression Regulation; Glycomics; Glycosylphosphatidylinositols; Heparitin Sulfate; Humans; Mannosyltransferases; Metabolic Networks and Pathways; Multiple Myeloma; Plasma Cells; Polysaccharides

This study was undertaken to generate human single chain variable antibody fragments (scFvs) reacting specifically against multiple myeloma (MM) cells using the phage display technique. To isolate myeloma-specific scFvs, we used a simple subtractive strategy by adsorbing the Griffin #1 antibody phage library against myeloma cells in the presence of excess decoy biotinylated HL60 cells, and then removing the unwanted decoy cells using streptavidin coated plates. From eleven scFvs that were isolated, two antibodies, D4A4 and D6B10 stained MM cell lines and patient MM cells with higher intensity than normal plasma cells. Both D4A4 and D6B10 scFvs immunoprecipitated syndecan-1 from myeloma cells and recognized sulfated motifs on syndecan-1-associated heparan sulfate (HS) chains. ScFv D4A4 competed with D6B10 for binding to MM cells. However, they differed in their fine specificities. ScFv D6B10 recognized HS 2,6-O-, N-sulfated motifs and, in contrast, binding of scFv D4A4 required N-sulfation combined with either 2-O- or 6-O-sulfation. Increased D6B10 binding on MM cells suggests that their HS chains contain a greater number of 2,6-O-, N-sulfated motifs than normal plasma cells. Since these highly sulfated motifs bind various angiogenic and growth factors and present them to their respective receptors, they could be instrumental for MM cell survival, proliferation and metastasis. Therefore, scFvs D4A4 and D6B10 provide a means to easily monitor changes in sulfation patterns of heparan sulfate during myeloma tumor progression.

Topics: Antibody Specificity; Antigens, Neoplasm; Cell Line, Tumor; Cell Surface Display Techniques; Heparitin Sulfate; HL-60 Cells; Humans; Multiple Myeloma; Plasma Cells; Single-Chain Antibodies; Syndecan-1

Fusion proteins containing protein transduction domain (PTD) are widely used for intracellular delivery of exogenous proteins. PTD-mediated delivery requires expression of heparan sulfate on the surface of the target cells. However, some of metastatic tumor cells and primary lymphocytes poorly express heparan sulfate. Here we demonstrate that proteins complexed with nanosize hydrogels formed by cationic cholesteryl group-bearing pullulans (cCHP) are efficiently delivered to myeloma cells and primary CD4(+) T lymphocytes probably by induction of macropinocytosis, although these cells are resistant to PTD-mediated protein delivery as a consequence of poor heparan sulfate expression. The anti-apoptotic protein Bcl-xL delivered by cCHP nanogels efficiently blocked apoptosis of these cells, establishing functional regulation of cells by proteins delivered by cCHP nanogels. Thus, cCHP nanogel is a useful tool to deliver proteins for development of new cancer therapy and immune regulation.

Topics: Animals; Cations; Cell Line, Tumor; Flow Cytometry; Heparitin Sulfate; Mice; Mice, Inbred C57BL; Multiple Myeloma; Nanostructures; Nanotechnology; T-Lymphocytes

Expression of the heparan sulfate proteoglycan syndecan-1 is a hallmark of both normal and multiple myeloma (MM) plasma cells. Syndecan-1 could affect plasma cell fate by strengthening integrin-mediated adhesion via its core protein and/or by accommodating and presenting soluble factors via its HS side chains. Here, we show that inducible RNAi-mediated knockdown of syndecan-1 in human MM cells leads to reduced growth rates and a strong increase of apoptosis. Importantly, knockdown of EXT1, a copolymerase critical for HS chain biosynthesis, had similar effects. Using an innovative myeloma xenotransplantation model in Rag-2(-/-)gamma(c)(-/-) mice, we demonstrate that induction of EXT1 knockdown in vivo dramatically suppresses the growth of bone marrow localized myeloma. Our findings provide direct evidence that the HS chains of syndecan-1 are crucial for the growth and survival of MM cells within the bone marrow environment, and indicate the HS biosynthesis machinery as a potential treatment target in MM.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; Doxycycline; Drug Delivery Systems; Gene Targeting; Heparitin Sulfate; Humans; Immunoglobulin gamma-Chains; Mice; Mice, Knockout; Multiple Myeloma; N-Acetylglucosaminyltransferases; RNA, Small Interfering; Syndecan-1; Xenograft Model Antitumor Assays

Heparanase enhances shedding of syndecan-1 (CD138), and high levels of heparanase and shed syndecan-1 in the tumor microenvironment are associated with elevated angiogenesis and poor prognosis in myeloma and other cancers. To explore how the heparanase/syndecan-1 axis regulates angiogenesis, we used myeloma cells expressing either high or low levels of heparanase and examined their impact on endothelial cell invasion and angiogenesis. Medium conditioned by heparanase-high cells significantly stimulated endothelial invasion in vitro compared with medium from heparanase-low cells. The stimulatory activity was traced to elevated levels of vascular endothelial growth factor (VEGF) and syndecan-1 in the medium. We discovered that the heparan sulfate chains of syndecan-1 captured VEGF and also attached the syndecan-1/VEGF complex to the extracellular matrix where it then stimulated endothelial invasion. In addition to its heparan sulfate chains, the core protein of syndecan-1 was also required because endothelial invasion was blocked by addition of synstatin, a peptide mimic of the integrin activating region present on the syndecan-1 core protein. These results reveal a novel mechanistic pathway driven by heparanase expression in myeloma cells whereby elevated levels of VEGF and shed syndecan-1 form matrix-anchored complexes that together activate integrin and VEGF receptors on adjacent endothelial cells thereby stimulating tumor angiogenesis.

Topics: Aorta; Cell Line, Tumor; Culture Media, Conditioned; Endothelium; Extracellular Matrix; Glucuronidase; Heparitin Sulfate; Humans; Multiple Myeloma; Neoplasm Invasiveness; Neovascularization, Pathologic; Organ Culture Techniques; Syndecan-1; Vascular Endothelial Growth Factor A

Syndecan-1 is a proteoglycan that concentrates heparin-binding factors on the surface of multiple myeloma cells, and probably plays a major role in multiple myeloma biology. As heparan sulphate and chondroitin sulphate are the bioactive components of syndecan-1, we analysed the signature of genes encoding 100 proteins involved in synthesis of these chains, i.e. from precursor uptake to post-translational modifications, using Affymetrix microarrays. The expression of enzymes required for heparan sulphate and chondroitin sulphate biosynthesis was shown to increase in parallel with syndecan-1 expression, throughout the differentiation of memory B cells into plasmablasts and normal bone marrow plasma cells. Sixteen genes were significantly different between normal and malignant plasma cells, nine of these genes -EXT2, CHSY3, CSGALNACT1, HS3ST2, HS2ST1, CHST11, CSGALNACT2, HPSE, SULF2 - encode proteins involved in glycosaminoglycan chain synthesis or modifications. Kaplan-Meier analysis was performed in two independent series of patients: B4GALT7, CSGALNACT1, HS2ST1 were associated with a good prognosis whereas EXT1 was linked to a bad prognosis. This study provides an overall picture of the major genes encoding for proteins involved in heparan sulphate and chondroitin sulphate synthesis and modifications that can be implicated in normal and malignant plasma cells.

Topics: B-Lymphocytes; Case-Control Studies; Cell Line, Tumor; Chondroitin Sulfates; Gene Expression; Gene Expression Profiling; Heparitin Sulfate; Humans; Immunologic Memory; Kaplan-Meier Estimate; Multiple Myeloma; Oligonucleotide Array Sequence Analysis; Plasma Cells; Principal Component Analysis; Prognosis; Protein Processing, Post-Translational; Syndecan-1

Among the four members of the syndecan family there exists a high level of divergence in the ectodomain core protein sequence. This has led to speculation that these core proteins bear important functional domains. However, there is little information regarding these functions, and thus far, the biological activity of syndecans has been attributed largely to their heparan sulfate chains. We have previously demonstrated that cell surface syndecan-1 inhibits invasion of tumor cells into three-dimensional gels composed of type I collagen. Inhibition of invasion is dependent on the syndecan heparan sulfate chains, but a role for the syndecan-1 ectodomain core protein was also indicated. To more closely examine this possibility and to map the regions of the ectodomain essential for syndecan-1-mediated inhibition of invasion, a panel of syndecan-1 mutational constructs was generated, and each construct was transfected individually into myeloma tumor cells. The anti-invasive effect of syndecan-1 is dramatically reduced by deletion of an ectodomain region close to the plasma membrane. Further mutational analysis identified a stretch of 5 hydrophobic amino acids, AVAAV (amino acids 222-226), critical for syndecan-1-mediated inhibition of cell invasion. This invasion regulatory domain is 26 amino acids from the start of the transmembrane domain. Importantly, this domain is functionally specific because its mutation does not affect syndecan-1-mediated cell binding to collagen, syndecan-1-mediated cell spreading, or targeting of syndecan-1 to specific cell surface domains. This invasion regulatory domain may play an important role in inhibiting tumor cell invasion, thus explaining the observed loss of syndecan-1 in some highly invasive cancers.

Topics: Amino Acid Sequence; Animals; Cell Line, Tumor; Heparitin Sulfate; Humans; Membrane Glycoproteins; Mice; Molecular Sequence Data; Multiple Myeloma; Mutagenesis, Site-Directed; Neoplasm Invasiveness; Protein Structure, Tertiary; Proteoglycans; Syndecan-1; Syndecans

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.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Disaccharides; Disease Progression; DNA, Complementary; Extracellular Matrix; Fibroblast Growth Factor 2; Flow Cytometry; Growth Substances; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Immunohistochemistry; Mice; Mice, SCID; Multiple Myeloma; Neoplasm Transplantation; Neoplasms; Polysaccharide-Lyases; Protein Binding; Signal Transduction; Sulfatases; Sulfotransferases; Time Factors; Transfection

Sperm protein 17 (Sp17) is a highly conserved mammalian protein present on acrosome-reacted sperm that is thought to promote fertilization by binding sulfated carbohydrates of the oocyte zona pellucida. Although Sp17 was originally described as a testis-specific antigen, emerging evidence indicates that it may be more ubiquitously expressed than was previously thought. With the use of a specific antiserum, Sp17 was found to be present on the surface of malignant lymphoid cells, including B- and T-lymphoid cell lines, and on the surface of primary cells isolated from 2 patients having B-lymphoid tumors. Surprisingly, circulating B lymphocytes isolated from healthy volunteers also expressed Sp17, while circulating T lymphocytes exhibited only very weak expression. The role of Sp17 in promoting lymphoid cell adhesion was addressed with the use of recombinant Sp17 (rSp17). The rSp17 binds to the surface of myeloma cells but not to cells pretreated with heparitinase, an enzyme that removes heparan sulfate from the cell surface. Moreover, rSp17 promotes extensive aggregation of cells that express the syndecan-1 heparan sulfate proteoglycan, but in contrast, cells lacking syndecan-1 expression fail to aggregate in the presence of rSp17. These findings suggest that Sp17 promotes heparan sulfate-mediated cell aggregation and thereby plays a role in regulating adhesion and migration of normal and malignant lymphocytes.

Topics: Antigens, Surface; Calmodulin-Binding Proteins; Carrier Proteins; Cell Adhesion; Cell Movement; Fluorescent Antibody Technique, Indirect; Heparitin Sulfate; Humans; Lymphocytes; Membrane Proteins; Multiple Myeloma; RNA, Messenger; Tumor Cells, Cultured

Two myeloma cell lines, MPC-11 and P3X63Ag8.653 (P3), have almost identical amounts of syndecan-1 at their cell surface. The syndecan-1 molecules from both lines are similar in size, have indistinguishable core proteins, and have similarly sized heparan sulfate chains. Nevertheless, syndecan-1 on MPC-11 mediates cell adhesion to type I collagen, whereas P3 cells do not bind collagen. Affinity co-electrophoresis reveals that intact syndecan-1 isolated from P3 cells binds collagen poorly and that syndecan-1 heparan sulfate isolated from MPC-11 has a 20-fold higher affinity for collagen than syndecan-1 heparan sulfate from P3. Analysis of disaccharide composition and oligosaccharide mapping also reveals differences between MPC-11 and P3 heparan sulfate. Most notably, the level of N-sulfation and 2-O-sulfation is higher, and 6-O-sulfation lower, in syndecan-1 heparan sulfate from MPC-11 than from P3. Interestingly, levels of total sulfation of syndecan-1 heparan sulfate from MPC-11 and P3 are similar (75.6 and 72.6 sulfates/100 disaccharides, respectively), indicating that the difference in their affinity for collagen is not due to a difference in net charge. These data indicate that the fine structure of heparan sulfate can differ on identical proteoglycan core proteins, and these differences can control fundamental cellular properties such as cell-matrix adhesion.

Topics: Animals; Binding Sites; Carbohydrate Conformation; Cell Adhesion; Collagen; Heparitin Sulfate; Humans; Mice; Multiple Myeloma; Structure-Activity Relationship; Tumor Cells, Cultured

The syndecans comprise a family of integral membrane proteoglycans that regulate cell behaviors by binding to extracellular matrix and binding growth factors. In mouse blood cells, syndecan expression is restricted to cells of the B-cell lineage where it is expressed by pre-B cells and plasma cells, but is absent from circulating B cells. In the present study, we examined the expression, structure, and function of syndecan on human myeloma cell lines and myeloma patient bone marrow cells. On myeloma cells, syndecan is a small (modal relative molecular mass [M(r)] = 120 Kd) heparan sulfate proteoglycan localized at the cell surface. Syndecan was detected by immunodot blotting on 7 of 10 human myeloma cell lines and by reverse transcriptase polymerase chain reaction on 10 of 14 patient samples. Cell binding assays show that myeloma cells expressing syndecan bind to type I collagen via heparan sulfate chains, while those cell lines not expressing syndecan do not bind to collagen. Furthermore, the cell lines expressing syndecan were negative for CD19 and CD45 staining, indicating that syndecan expression is restricted to tumors having a well-differentiated phenotype. We conclude that syndecan acts as a matrix receptor on human myeloma cells but is not expressed by all tumors, suggesting that syndecan may participate in regulating myeloma cell adhesion to the bone marrow stromal matrix.

Topics: Cell Adhesion; Chromatography, Ion Exchange; Collagen; Electrophoresis, Polyacrylamide Gel; Flow Cytometry; Gene Expression; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Immunoblotting; Membrane Glycoproteins; Molecular Weight; Multiple Myeloma; Phenotype; Proteoglycans; RNA, Messenger; Syndecans; Tumor Cells, Cultured

We have identified a circulating, heparin-like anticoagulant in a patient with multiple myeloma (IgG4 lambda) who had serious clinically evident bleeding that contributed to his death. Purification of the patient's circulating coagulation inhibitor was accomplished by ammonium sulfate concentration, anion exchange chromatography, and affinity chromatography on protamine sulfate. Analysis of the purified inhibitor showed that it was a proteoglycan that comigrated with heparan sulfate on lithium acetate-agarose-gel electrophoresis and that it contained 39 per cent L-iduronic acid. Control samples of heparan sulfate and heparin contained 29 and 68 per cent L-iduronic acid, respectively. Functional coagulation studies revealed that the purified inhibitor had cofactor activity with antithrombin III that could be abolished by prior incubation with protamine sulfate or platelet factor 4. Recognition of the existence of this or of other similar inhibitors in bleeding patients is important because of the potential for treatment with agents such as protamine sulfate and platelet factor 4, which neutralize the anticoagulant effects of proteoglycans.

Topics: Blood Coagulation; Blood Coagulation Tests; Chromatography, Affinity; Chromatography, Ion Exchange; Electrophoresis, Agar Gel; Glycosaminoglycans; Hemorrhage; Heparitin Sulfate; Humans; Iduronic Acid; Male; Middle Aged; Multiple Myeloma

Heparan sulfate glycosaminoglycan, isolated from the cell surface of nonadhering murine myeloma cells (P3X63-Ag8653), does not bind to plasma fibronectin, but binds partially to collagen type I, as assayed by affinity chromatography with proteins immobilized on cyanogen bromide-activated Sepharose 4B. Identical results were obtained when myeloma heparan sulfate was cochromatographed, on the same fibronectin and collagen columns, with cell surface heparan sulfates collagen columns, with cell surface heparan sulfates from adhering Swiss mouse 3T3 and SV3T3 cells. These latter heparan sulfates do, however, bind to both fibronectin and collagen, as reported earlier (Stamatoglou, S.C., and J.M. Keller, 1981, Biochim. Biophys. Acta., 719:90-97). Cell adhesion assays established that hydrated collagen substrata can support myeloma cell attachment, but fibronectin cannot. Saturation of the heparan sulfate binding sites on the collagen substrata with heparan sulfate or heparin, prior to cell inoculation, abolished the ability to support cell adhesion, whereas chondroitin 4 sulfate, chondroitin 6 sulfate, and hyaluronic acid had no effect.

Topics: Animals; Cell Adhesion; Cell Line; Collagen; Fibronectins; Glycosaminoglycans; Heparitin Sulfate; Humans; Mice; Multiple Myeloma; Surface Properties

A woman, aged 68, with multiple myeloma (immunoglobulin[Ig]A kappa type) developed an anticoagulant with properties suggestive of heparin. The anticoagulant prolonged the thrombin time but not the reptilase time and was resistant to boiling, proteolytic enzyme digestion, and trichloracetic acid precipitation. The thrombin time was corrected by the addition (in vitro) of protamine sulfate or the addition of purified platelet Factor 4 (PF4) to the plasma. The anticoagulant was isolated by PF4-Sepharose affinity chromatography and analyzed in terms of its molecular weight, uronic acid, and amino acid composition. The proteoglycan isolated had a mol wt of 116,000 and appears to consist of two 38,000 dalton polysaccharide units interconnected by peptide material totaling 39,000 daltons. Electrophoretic analysis of the pronase digested peptidoglycan using the lithium acetate-agarose technique suggested the material was of the heparan sulfate type. The peptidoglycan had about one-tenth the specific activity of commercially available heparin on a weight basis. The isolated proteoglycan was indistinguishable from commercial heparin when analyzed in terms of its ability to act as a cofactor in the antithrombin III inhibition of thrombin.

Topics: Aged; Amino Acids; Blood Coagulation; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Molecular Weight; Multiple Myeloma; Proteoglycans