Heparan sulfate proteoglycans and heparin regulate melanoma cell functions

Background

The solid melanoma tumor consists of transformed melanoma cells, and the associated stromal cells including fibroblasts, endothelial cells, immune cells, as well as, soluble macro- and micro-molecules of the extracellular matrix (ECM) forming the complex network of the tumor microenvironment. Heparan sulfate proteoglycans (HSPGs) are an important component of the melanoma tumor ECM. Importantly, there appears to be both a quantitative and a qualitative shift in the content of HSPGs, in parallel to the nevi–radial growth phase–vertical growth phase melanoma progression. Moreover, these changes in HSPG expression are correlated to modulations of key melanoma cell functions.

Scope of review

This review will critically discuss the roles of HSPGs/heparin in melanoma development and progression.

Major conclusions

We have correlated HSPGs' expression and distribution with melanoma cell signaling and functions as well as angiogenesis.

General significance

The current knowledge of HSPGs/heparin biology in melanoma provides a foundation we can utilize in the ongoing search for new approaches in designing anti-tumor therapy. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Functions of heparan sulfate proteoglycans in cell signaling during development

Heparan sulfate proteoglycans (HSPGs) are cell-surface and extracellular matrix macromolecules that are composed of a core protein decorated with covalently linked glycosaminoglycan (GAG) chains. In vitro studies have demonstrated the roles of these molecules in many cellular functions, and recent in vivo studies have begun to clarify their essential functions in development. In particular, HSPGs play crucial roles in regulating key developmental signaling pathways, such as the Wnt, Hedgehog, transforming growth factor-beta, and fibroblast growth factor pathways. This review highlights recent findings regarding the functions of HSPGs in these signaling pathways during development.     

Developmental roles of heparan sulfate proteoglycans in Drosophila

The formation of complex patterns in multi-cellular organisms is regulated by a number of signaling pathways. In particular, the Wnt and Hedgehog (Hh) pathways have been identified as critical organizers of pattern in many tissues. Although extensive biochemical and genetic studies have elucidated the central components of the signal transduction pathways regulated by these secreted molecules, we still do not understand fully how they organize gradients of gene activities through field of cells. Studies in Drosophila have implicated a role for heparan sulfate proteoglycans (HSPGs) in regulating the signaling activities and distribution of both Wnt and Hh. Here we review these findings and discuss various models by which HSPGs regulate the distributions of Wnt and Hh morphogens. Published in 2003.     

Upregulation of Glypicans in Hippo mutants alters the coordinated activity of morphogens

The function of the conserved Drosophila Hippo signaling pathway has been shown to be required to limit cell proliferation. Several studies have identified different target genes of this pathway that could modulate this function. However, the ectopic expression of these genes cannot account for all of the hyperplasic and pattern defects displayed by Hippo signaling mutants. We have recently identified two new targets of the Hippo pathway, the heparan sulfate proteoglycans (HSPGs) encoded by division abnormally delayed (dally) and dally-like protein (dlp). The function of these glypicans is required to modulate the activity of different signaling pathways triggered by diffusable ligands. Thus, our results link the function of the Hippo pathway with the control of the activity of several signaling pathways required for the definition of the size and pattern of an organ.     

Role of heparan sulfate proteoglycans in cell-cell signaling in Drosophila.

Heparan sulfate proteoglycans (HSPGs) are abundant molecules associated with the cell surface and extracellular matrix, and consist of a protein core to which heparan sulfate (HS) glycosaminoglycan (GAG) chains are attached. Although these molecules have been the focus of intense biochemical studies in vitro, their biological functions in vivo were unclear until recently. We have undertaken an in vivo functional study of HSPGs in Drosophila. Our studies, as well as others, demonstrate the critical roles of HSPGs in several major signaling pathways, including ibroblast growth factor (FGF), Wnt, Hedgehog (Hh) and TGF-beta. Our results also suggest that specific HS GAG chain modifications, as well as specific HSPG protein cores, are involved in specific signaling pathways.     

Heparin activates Wnt signaling for neuronal morphogenesis

Wnt factors are secreted ligands that affect different aspects of the nervous system behavior like neurodevelopment, synaptogenesis and neurodegeneration. In different model systems, Wnt signaling has been demonstrated to be regulated by heparan sulfate proteoglycans (HSPGs). Whether HSPGs modulate Wnt signaling in the context of neuronal behavior is currently unknown. Here we demonstrate that activation of Wnt signaling with the endogenous ligand Wnt-7a results in an increased of neurite outgrowth in the neuroblastoma N2a cell line. Interestingly, heparin induces glycogen synthase kinase-3beta (GSK-3beta) inhibition, beta-catenin stabilization and morphological differentiation in both N2a cells and in rat primary hippocampal neuronal cultures. We also show that heparin modulates Wnt-3a-induced stabilization of beta-catenin. Several extracellular matrix and membrane-attached HSPGs were found to be expressed in both in vitro neuronal models. Changes in the expression of specific HSPGs were observed upon differentiation of N2a cells. Taken together, our findings suggest that HSPGs may modulate canonical Wnt signaling for neuronal morphogenesis.     

Heparan sulfate proteoglycans (HSPGs) modulate BMP2 osteogenic bioactivity in C2C12 cells

Cell surface heparan sulfate proteoglycans (HSPGs) have been implicated in bone morphogenetic protein (BMP)-mediated morphogenesis by regulating BMP activity and gradient formation. However, the direct role of HSPGs in BMP signaling is poorly understood. Here we show that HSPGs directly regulate BMP2-mediated transdifferentiation of C2C12 myoblasts into osteoblasts. HSPGs sequester BMP2 at the cell surface and mediate BMP2 internalization. Depletion of cell surface HSPGs by heparinase III treatment or decreased glycosaminoglycan chain sulfation with sodium chlorate enhances BMP2 morpho-genetic bioactivity. The addition of exogenous heparin, a widely used anticoagulant, reduced BMP2 signaling. Our results suggest that cell surface HSPGs mediate BMP2 internalization and modulate BMP2 osteogenic activity.     

Functional binding of secreted molecules to heparan sulfate proteoglycans in Drosophila

Heparan sulfate proteoglycans (HSPGs) are associated with the cell surface and covalently linked to a small number of long unbranched chains of repeating disaccharides. Numerous biochemical studies of these extracellular matrix molecules have implicated them in a variety of biological phenomena, in particular cell-cell interactions. Recent genetic studies in Drosophila have begun to clarify the function of HSPGs in vivo and recent findings have implicated HSPGs in Wnt, Hedgehog, fibroblast growth factor and transforming growth factor-beta signaling pathways during development.     

When sugars guide axons: insights from heparan sulphate proteoglycan mutants

Although there have previously been hints that heparan sulphate proteoglycans (HSPGs) are important for axon guidance, as they are for many other biological processes, there has been little in vivo evidence for interaction with known axon-guidance pathways. Genetic analyses of fly, mouse, nematode and zebrafish mutants now confirm the role of HSPGs in axon guidance and are beginning to show that they might have a key role in modulating the action of axon-guidance ligands and receptors.     

Hear the Wnt Ror: how melanoma cells adjust to changes in Wnt

The interplay between canonical and non‐canonical Wnt pathways in development and tumorigenesis is tightly regulated. In this review we will describe the yin and the yang of canonical and non‐canonical Wnt signaling pathways during melanocyte development, and melanoma genesis. Canonical Wnt signaling, represented by Wnts such as Wnt1 and Wnt3A, signals via β‐catenin to promote melanocyte differentiation and tumor development. Non‐canonical Wnt signaling, specifically Wnt5A, regulates canonical pathways, and signals to induce melanoma metastasis. This review will focus on the role of Wnt5A during melanoma progression, and its relationship to canonical Wnt signaling.     

Roles of heparan sulfate sulfation in dentinogenesis

Cell surface heparan sulfate (HS) is an essential regulator of cell signaling and development. HS traps signaling molecules, like Wnt in the glycosaminoglycan side chains of HS proteoglycans (HSPGs), and regulates their functions. Endosulfatases Sulf1 and Sulf2 are secreted at the cell surface to selectively remove 6-O-sulfate groups from HSPGs, thereby modifying the affinity of cell surface HSPGs for its ligands. This study provides molecular evidence for the functional roles of HSPG sulfation and desulfation in dentinogenesis. We show that odontogenic cells are highly sulfated on the cell surface and become desulfated during their differentiation to odontoblasts, which produce tooth dentin. Sulf1/Sulf2 double null mutant mice exhibit a thin dentin matrix and short roots combined with reduced expression of dentin sialophosphoprotein (Dspp) mRNA, encoding a dentin-specific extracellular matrix precursor protein, whereas single Sulf mutants do not show such defective phenotypes. In odontoblast cell lines, Dspp mRNA expression is potentiated by the activation of the Wnt canonical signaling pathway. In addition, pharmacological interference with HS sulfation promotes Dspp mRNA expression through activation of Wnt signaling. On the contrary, the silencing of Sulf suppresses the Wnt signaling pathway and subsequently Dspp mRNA expression. We also show that Wnt10a protein binds to cell surface HSPGs in odontoblasts, and interference with HS sulfation decreases the binding affinity of Wnt10a for HSPGs, which facilitates the binding of Wnt10a to its receptor and potentiates the Wnt signaling pathway, thereby up-regulating Dspp mRNA expression. These results demonstrate that Sulf-mediated desulfation of cellular HSPGs is an important modification that is critical for the activation of the Wnt signaling in odontoblasts and for production of the dentin matrix.     

Heparan sulfate proteoglycans in Drosophila neuromuscular development

Heparan sulfate proteoglycans (HSPGs) are glycoconjugates bearing heparan sulfate (HS) chains covalently attached to core proteins, which are ubiquitously distributed on the cell surface and in the extracellular matrix. HSPGs interact with a number of molecules mainly through HS chains, which play critical roles in diverse physiological and disease processes. Among these, recent vertebrate studies showed that HSPGs are closely involved in synapse development and function. However, the detailed molecular mechanisms remain elusive. Genetic studies from fruit flies, Drosophila melanogaster, have begun to reveal the molecular mechanisms by which HSPGs regulate synapse formation at neuromuscular junctions (NMJs). In this review, we introduce Drosophila studies showing how HSPGs regulate various signaling pathways in developing NMJs. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.     

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.     

Sugar codes for axons?

Proteoglycans are complex macromolecules with the potential for extraordinary diversity. Several recent studies have demonstrated important roles for heparan sulfate and chondroitin sulfate proteoglycans (HSPGs and CSPGs) in axon pathfinding and have linked HSPGs to specific signaling pathways. More speculatively, there are hints of a "sugar code," in which specific sugar modifications might act instructively in guidance decisions. This raises the intriguing possibility that the complexity of neuronal wiring may in part reflect the complexity of proteoglycan modifications.     

The PTEN–AKT3 signaling cascade as a therapeutic target in melanoma

Melanocytes undergo extensive genetic changes during transformation into aggressive melanomas. These changes deregulate genes whose aberrant activity promotes the development of this disease. The phosphoinositide‐3‐kinase (PI3K) and mitogen‐activated protein (MAP) kinase pathways are two key signaling cascades that have been found to play prominent roles in melanoma development. These pathways relay extra‐cellular signals via an ordered series of consecutive phosphorylation events from cell surface throughout the cytoplasm and nucleus regulating diverse cellular processes including proliferation, survival, invasion and angiogenesis. It is generally accepted that therapeutic agents would need to target these two pathways to be an effective therapy for the long‐term treatment of advanced‐stage melanoma patients. This review provides an overview of the PI3 kinase pathway focusing specifically on two members of the pathway, called PTEN and Akt3, which play important roles in melanoma development. Mechanisms leading to deregulation of these two proteins and therapeutic implications of targeting this signaling cascade to treat melanoma are detailed in this review.     

Heparan sulphate proteoglycans: the sweet side of development

Pattern formation during development is controlled to a great extent by a small number of conserved signal transduction pathways that are activated by extracellular ligands such as Hedgehog, Wingless or Decapentaplegic. Genetic experiments have identified heparan sulphate proteoglycans (HSPGs) as important regulators of the tissue distribution of these extracellular signalling molecules. Several recent reports provide important new insights into the mechanisms by which HSPGs function during development.     

QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling

The 6-O sulfation states of cell surface heparan sulfate proteoglycans (HSPGs) are dynamically regulated to control the growth and specification of embryonic progenitor lineages. However, mechanisms for regulation of HSPG sulfation have been unknown. Here, we report on the biochemical and Wnt signaling activities of QSulf1, a novel cell surface sulfatase. Biochemical studies establish that QSulf1 is a heparan sulfate (HS) 6-O endosulfatase with preference, in particular, toward trisulfated IdoA2S-GlcNS6S disaccharide units within HS chains. In cells, QSulf1 can function cell autonomously to remodel the sulfation of cell surface HS and promote Wnt signaling when localized either on the cell surface or in the Golgi apparatus. QSulf1 6-O desulfation reduces XWnt binding to heparin and HS chains of Glypican1, whereas heparin binds with high affinity to XWnt8 and inhibits Wnt signaling. CHO cells mutant for HS biosynthesis are defective in Wnt-dependent Frizzled receptor activation, establishing that HS is required for Frizzled receptor function. Together, these findings suggest a two-state "catch or present" model for QSulf1 regulation of Wnt signaling in which QSulf1 removes 6-O sulfates from HS chains to promote the formation of low affinity HS-Wnt complexes that can functionally interact with Frizzled receptors to initiate Wnt signal transduction.