Báo cáo khoa học: Proteoglycans in health and disease: novel roles for proteoglycans in malignancy and their pharmacological targeting

M I N I R E V I E W

Proteoglycans in health and disease: novel roles for proteoglycans in malignancy and their pharmacological targeting Achilleas D. Theocharis1, Spyridon S. Skandalis2, George N. Tzanakakis3 and Nikos K. Karamanos1

1 Laboratory of Biochemistry, Department of Chemistry, University of Patras, Greece 2 Ludwig Institute for Cancer Research Ltd, Uppsala, Sweden 3 Department of Histology, University of Crete, Heraklion, Greece

Keywords cancer; decorin; glypicans; lumican; molecular targeting; perlecan; proteoglycans; serglycin; syndecans; versican

Correspondence N. Karamanos, Laboratory of Biochemistry, Department of Chemistry, University of Patras, 26100 Patras, Greece Fax: +30 2610 997153 Tel: +30 2610 997915 E-mail: n.k.karamanos@upatras.gr

(Received 3 May 2010, revised 15 July 2010, accepted 29 July 2010)

doi:10.1111/j.1742-4658.2010.07800.x

The expression of proteoglycans (PGs), essential macromolecules of the tumor microenvironment, is markedly altered during malignant transforma- tion and tumor progression. Synthesis of stromal PGs is affected by factors secreted by cancer cells and the unique tumor-modiﬁed extracellular matrix may either facilitate or counteract the growth of solid tumors. The emerg- ing theme is that this dual activity has intrinsic tissue speciﬁcity. Matrix- accumulated PGs, such as versican, perlecan and small leucine-rich PGs, affect cancer cell signaling, growth and survival, cell adhesion, migration and angiogenesis. Furthermore, expression of cell-surface-associated PGs, such as syndecans and glypicans, is also modulated in both tumor and stro- mal cells. Cell-surface-associated PGs bind various factors that are involved in cell signaling, thereby affecting cell proliferation, adhesion and motility. An important mechanism of action is offered by a proteolytic processing of cell-surface PGs known as ectodomain shedding of syndecans; this facili- tates cancer and endothelial cell motility, protects matrix proteases and provides a chemotactic gradient of mitogens. However, syndecans on stro- mal cells may be important for stromal cell ⁄ cancer cell interplay and may promote stromal cell proliferation, migration and angiogenesis. Finally, abnormal PG expression in cancer and stromal cells may serve as a bio- marker for tumor progression and patient survival. Enhanced understand- ing of the regulation of PG metabolism and the involvement of PGs in cancer may offer a novel approach to cancer therapy by targeting the tumor microenvironment. In this minireview, the implication of PGs in cancer development and progression, as well as their pharmacological targeting in malignancy, are presented and discussed.

Introduction

Proteoglycans (PGs) are macromolecules composed of a speciﬁc core protein substituted with covalently

linked glycosaminoglycan (GAG) chains named chon- droitin sulfate (CS), dermatan sulfate (DS), keratan

Abbreviations ADAMTS, a disintegrin and metalloprotease domain with thrombospondin motifs; CS, chondroitin sulfate; DS, dermatan sulfate; ECM, extracellular matrix; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FGF, ﬁbroblast growth factor; GAG, glycosaminoglycan; HA, hyaluronan; HC, hepatocellular carcinoma; Hh, hedgehog; HS, heparan sulfate; HSPGs, heparan sulfate proteoglycans; MMP, matrix metalloproteinases; PDGF, platelet-derived growth factor; PG, proteoglycan; SLRPs, small leucine-rich proteoglycans; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.

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their potential

as pharmacological

sulfate, heparin and heparan sulfate (HS). Hyaluronan (HA) is the only GAG synthesized in a free form not covalently bound on a core protein. GAGs are linear, negatively charged polysaccharides comprised of repeating disaccharides of acetylated hexosamines (N-acetyl-galactosamine or N-acetyl-glucosamine) and mainly by uronic acids (d-glucoronic acid or l-iduron- ic acid) being sulfated at various positions. Keratan sulfate is the only GAG to be comprised of repeating disaccharides containing N-acetyl-glucosamine and galactose.

Structural modiﬁcations of GAGs may facilitate tumorigenesis in various ways, modulating the func- tions of PGs. Our rapid increase in knowledge that PGs are among the key players in the tumor microen- vironment and can modulate tumor progression, sug- gests targets. Pharmacological treatment may target PG metabolism, their utilization as targets for immunotherapy or their direct use as therapeutic agents. In this minireview, we focus on the roles of PG in cancer development and progression, as well as their pharmacological targeting in malignancy.

Extracellular matrix PGS

further

Hyalectans

The subfamily of hyalectans includes versican, aggre- can, neurocan and brevican. Hyalectans have the abil- ity to bind HA through their N-terminal globular domain (G1), the central domain carries most of the GAG chains and exhibits lectin-like activity located in the C-terminal globular domain (G3) (Fig. 1) [1].

in cancer and

Versican expression and functions inﬂammation

functions acting as

Versican is expressed throughout the body and pro- vides ECM with hygroscopic properties creating a loose and hydrated matrix that is necessary to support key events in development and disease. Four splice- variants of human versican (V0, V1, V2 and V3) have been identiﬁed. The differences among the versican splice-variants are found in the central portion of the protein core, and they vary in the number and

PGs can be classiﬁed into three main groups accord- ing to their localization, extracellularly secreted, those associated with the cell surface and intracellular. Each main group is classiﬁed into subfamilies according to their gene homology, core protein proper- ties, size and modular composition. Secreted PGs involve large aggregating PGs, named hyalectans, small leucine-rich PGs (SLRPs) and basement mem- brane PGs. Cell-surface-associated PGs are divided into two main subfamilies (syndecans and glypicans), whereas serglycin is the only intracellular PG charac- terized to date [1,2]. The wide molecular diversity of PGs derives from the multitude of possible combina- tions of protein cores, O-linked and N-linked oligosac- charides, and various types and numbers of GAG chains. The speciﬁc structural characteristics of GAG types provide some of the structural basis for the mul- titude of their biological functions [3]. PGs exhibit structural numerous biological components in tissue organization, and affect several cellular parameters, such as cell proliferation, adhe- sion, migration and differentiation. PGs interact with growth factors and cytokines, as well as with growth factor receptors, and are implicated in cell signaling. The catalog of physiological functions and the patho- biological roles in which PGs are involved have grown rapidly.

Hyalectans and SLRPs

Hyaluronan

GAG chains

EGF like motifs

Versican

Lectin-like repeat

Complement regulatory protein

Aggrecan

Collagen I

Neurocan

Brevican

SLRPs

Fig. 1. Schematic representation of hyalectans (hyaluronan binding PGs) and SLRPs found in ECM.

During carcinogenesis, malignant cells secrete solu- ble growth factors that stimulate cell growth and acti- vate stromal cells to secrete effectors that in turn stimulate further tumor cell growth. Both activated stromal and tumor cells are implicated in the reorgani- zation of the extracellular matrix (ECM) to facilitate tumor cell growth, migration and invasion. PG expres- sion is markedly modiﬁed in the tumor microenviron- ment. Altered expression of PGs on tumor and stromal cell membranes affects cancer cell signaling, growth and survival, cell adhesion, migration and angiogenesis. The type and ﬁne structure of GAG chains attached to PGs are markedly affected in the context of malignant transformation as result of the altered expression of GAG-synthesizing enzymes.

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thrombospondin motifs (ADAMTS), matrix metallo- proteinases (MMPs) and plasmin can cleave versican generating fragments containing the G1 domain in some cases [4,5]. Thus, regulation of G1 and G3 versi- can levels by proteases is an important factor in cancer cell motility and metastasis (Table 1).

presence of GAGs. Versican is able to regulate many cellular processes including adhesion, proliferation, apoptosis, migration, invasion and ECM assembly via the highly negatively charged CS ⁄ DS side chains, and by interactions of the G1 and G3 domains with other proteins [4–6]. Versican binds to ECM components such as HA, type I collagen, tenascin-R, ﬁbulin-1 and -2, ﬁbrillin-1, ﬁbronectin, P- and L-selectin and chemokin- es. Versican also binds to the cell-surface proteins CD44, integrin b1, epidermal growth factor receptor (EGFR), P-selectin glycoprotein ligand-1 [6,7] and toll- like receptor 2 [8].

of malignancies.

Indeed,

Versican may also promote the formation of an inﬂammatory microenvironment in the tumor stroma. The interaction between versican and toll-like receptor- 2 links inﬂammation and metastasis [8,32]. Ligation of toll-like receptor-2 present on endothelial cells and ﬁbroblasts by versican activates these cells and triggers the secretion of inﬂammatory cytokines. This is mecha- nistically important insofar as inﬂammation is often associated with cancer initiation and promotion, and inﬂammatory cells (primarily macrophages) are consis- tently regarded as critical mediators in the develop- ment tumor-associated macrophages can enhance angiogenesis, ECM degrada- tion and remodeling, and can promote cancer cell invasion [32].

types

Notably, the versican gene promoter contains a p53- binding site in its ﬁrst intron and can be directly acti- vated by the tumor suppressor p53 in a dose-depen- dent manner [33,34]. It was found to be a target gene of Wnt signaling in human embryonic carcinoma cells [35].

Versican regulation and targeting

V0 and V1 are the predominant isoforms present in cancer tissues [9–12]. Overexpression of the V3 isoform in melanoma cells markedly reduces cell growth in vitro and in vivo [13], and promotes pulmonary metastases [14]. V3 isoform may have a dual role as an inhibitor of tumor growth and a stimulator of metasta- sis. Elevated levels of versican have been reported in most malignancies to date and have been associated with cancer relapse and poor patient outcome in breast, [10,15–21] prostate and many other cancer (Table 1). We have seen that versican is accumulated in the preclinical phase of breast cancer in nonpalpable breast carcinomas (Lambropoulou et al., personal data). This accumulation seems to be associated with risk factors because increased mammographic density and malignant appearing microcalciﬁcations are found in these cases. Versican appears to be most commonly secreted by the activated peritumoral stromal cells in adenocarcinomas [20,21]. However, human pancreatic cancer cells can also secrete versican [22].

to ﬁbronectin-coated surfaces

reducing this powerful

several protease families

Functional studies have demonstrated that versican can increase cancer cell motility [12,23–25], prolifera- tion [26] and metastasis [27]. Cancer cells can form a polarized pericellular sheath through compartmental- ized cell-surface CD44 expression and subsequent assembly of HA ⁄ versican aggregates that promotes their motility (Fig. 2) [25]. Soluble versican is able to reduce the attachment of prostate cancer and mela- in vitro noma cells [28,29]. Both the G1 and G3 versican domains have been shown to promote cell proliferation in NIH-3T3 ﬁbroblasts and tumor cells [23,24,30,31] (Table 1). The G1 domain of versican stimulates proliferation by destabilizing cell adhesion, whereas the G3 domain induces proliferation, at least in part, by activating EGFR via the action of epidermal growth factor (EGF)-like motifs (Fig. 2). G1 and G3 domains may differentially control tumor growth rate and have interactive roles to promote tumor development and metastasis. Notably, that include a disintegrin and metalloprotease domain with

Matrix effectors including platelet-derived growth fac- tor (PDGF), transforming growth factor b, interleukin 1b, angiotensin II and steroid hormones affect versican synthesis in various normal cell lines [6]. Transforming growth factor b has been found to regulate synthesis of versican in ﬁbrosarcoma, osteosarcoma and glioma cells [11,12,36]. The expression of versican can be also insulin-like growth factor I and modiﬁed by EGF, PDGF-BB in malignant mesothelioma cells [37]. The effect of protein tyrosine kinase signaling pathways on versican synthesis can be reversed following treatment with various tyrosine kinase inhibitors [38]. Therefore, targeting versican synthesis may be a potential mecha- nism for tumor-promoting agent. In agreement with this concept, the tyrosine kinase inhibitor genistein can block versican expression induced by growth factors in malignant mesothelioma cell lines [37]. Genetic and preclinical studies support the targeting of growth factor [transforming growth factor b, PDGF, EGF and vascular endothelial growth factor (VEGF)] signaling as a therapeutic strategy for combating cancer. To date, several approaches to inhi- bit growth factor signaling pathways in cancer have

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Table 1. Expression of proteoglycans in malignancies and possible pharmacological targeting. ADAMTS, a disintegrin and metalloprotease domain with thrombospondin motifs; HA, hyaluronan; MMP, matrix metalloproteinases.

Possibilities for potential pharmacological targeting

Proteoglycan subfamilies

Expression in malignancies ⁄ roles

Hyalectans Versican

› Brain tumors, melanomas, osteosarcomas, lymphomas, acute monocytic leukemia, testicular tumors, breast, prostate, colon, lung, pancreatic, endometrial, ovarian and oral cancers

Prognostic factor, proliferation, adhesion, motility,

metastasis, angiogenesis

Inhibition of synthesis (a) Blocking of growth factors; blocking antibodies, kinase inhibitors, antisense oligonucleotides, (b) miRNA-based therapeutics; miR-199a* Inhibition of degradation (a) ADAMTS, (b) MMPs inhibitors Inhibition of interaction with HA (a) HA oligomers

Aggrecan

Brevican

ﬂ Laryngeal cancer Cartilage degradation › Glioma Diagnostic marker, adhesion, motility

Inhibition of cleavage (a) ADAMTS, (b) MMPs inhibitors Immunotherapy

SLRPs Decorin

› Osteosarcoma, testicular tumors, ovarian, colon, gastric, pancreatic, laryngeal, breast cancer

Upregulation of synthesis (a) Viral-mediated delivery, (b) Demethylation agents, (c) Proteasome inhibitors Admistration of decorin protein or synthetic peptides

Lumican

Prognostic factor, inhibition of growth and migration (ﬂ ErbB and Met signaling) › Osteosarcoma, melanoma, breast cancer Prognostic factor, inhibition of growth and migration

Upregulation of synthesis (a) Viral-mediated delivery Admistration of lumican protein or synthetic peptides

Basement membranes Collagen XVIII

Admistration of endostatin fragment or synthetic peptides Endostatin producing cells in immunoisolation devices

Perlecan

Admistration of endorepellin fragment or synthetic peptides

Agrin

› Ovarian, pancreatic cancer ﬂ Liver and oral cancer Promoter of growth and angiogenesis Inhibitor of angiogenesis (endostatin) › Liver, oral tumors, melanoma Promoter of growth and angiogenesis Inhibitor of angiogenesis (endorepellin) › Liver tumors. Promoter of growth and angiogenesis? Inhibitor of angiogenesis (C-terminus)?

Admistration of C-terminal fragment or synthetic peptides

Cell surface Glypican-1

› Breast, pancreatic cancer Proliferation, growth factor signaling

Glypican-3

Inhibition of synthesis (a) Blocking of growth factors; blocking Abs, kinase inhibitors, antisense oligonucleotides, (b) siRNA-based therapeutics Immunotherapy Admistration of soluble fragments Immunotherapy

ﬂ Lung, gastric, ovarian, breast cancer and mesothelioma Inhibition of proliferation and induction of apoptosis › Neuroblastoma, Wilm’s tumor, hepatocellular carcinoma, melanoma

Syndecan-1

Immunotherapy Heparanase inhibitors

Promoter of tumor cell growth › Breast, prostate, head and neck, myeloma ﬂ Lung, colorectal, endometrial, cervical gastric, pancreatic cancer

Prognostic factor, proliferation, adhesion, migration,

Syndecan-2

Immunotherapy Heparanase inhibitors

differentiation, angiogenesis › Lung, ovarian, osteosarcoma, brain tumors, colon, mesothelioma

Syndecan-4

Proliferation, adhesion, migration, angiogenesis › Breast cancer, melanoma Adhesion and migration

Immunotherapy Heparanase inhibitors

Intracellular Serglycin

› Hematological malignancies Diagnosis

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Biological roles of versican

expression system, may be useful for targeting versican among the other genes involved in tumorigenesis [41] (Table 1).

Versican

Hyaluronan

EGFR

G3 domain

CD44

P

EGFR signaling Reduced cell adhesion

Tumor / endothelial cell proliferation and motility

future

The use of an antibody against to the ADAMTS- speciﬁc versican cleavage site inhibits glioma cell migration in a dose-dependent manner, suggesting that the local accumulation of versican fragments may also promote cancer cell motility and invasion (Fig. 2) [12]. Concurrent with this hypothesis, processed versican fragments have been identiﬁed in the peritumoral stroma of prostate cancer in association with higher ADAMTS1 and ADAMTS4 levels, but not in the interface surrounding normal prostate glands where full-legth versican is present [42]. Notably, the broad- spectrum MMP inhibitor GM6001 (Galardin), which inhibits the activity of MMPs and ADAMTS prote- invasion ases, has been shown to inhibit cancer cell and metastasis in a transgenic breast cancer model [43]. Other protease inhibitors such as catechin gallate esters, present in natural sources (green tea) have been shown to selectively inhibit ADAMTS-1, -4 and -5 and aggrecan catabolism in cartilage [44]. The ability of MMPs and ADAMTS inhibitors to prevent versican catabolism and versican-induced motility and metasta- sis may be an interesting area of study (Table 1).

Manipulation of

Fig. 2. Versican interacts with hyaluronan creating large aggre- gates. CD44 interacts with HA and versican forming a polarized and highly hydrated pericellular sheath that destabilizes cell adhe- sion and promotes cell motility. These aggregates facilitate further cell-shape changes and promote cell proliferation. EGF-like motifs present in the G3 domain of versican activate EGFR promoting cell proliferation and motility.

there are no data to show that

synthesis

regulated

the versican catabolic pathways may also provide novel therapeutic targets for cancer invasion and metastasis. For example, formation of a pericellular matrix rich in HA and versican implicated in cancer cell motility, could be inhibited by treatment with HA oligomers. Disruption of the HA–CD44 interaction with HA oligomers has been shown to markedly inhibit the growth of B16F16 melanoma cells [45]. In addition, HA oligomers inhibit the formation of receptor tyrosine kinases complexes and their phos- phorylation in prostate, colon and breast carcinoma cells [46]. Thus, the use of HA oligomers is a poten- tially attractive agent to block the formation of large versican–HA aggregates and HA–CD44 interactions, as well as local tumor invasion (Table 1).

Aggrecan degradation during cartilage destruction

been investigated. These approaches mainly include: (a) inhibition at the translational level using antisense oligonucleotides that can be engineered into immune cells or delivered directly into tumors, (b) inhibition of the ligand–receptor interaction using monoclonal anti- bodies, and (c) inhibition of the receptor-mediated sig- naling cascade using speciﬁc tyrosine kinase inhibitor. However, such approaches are effective in inhibiting the effects of versican in cancer cell models (Table 1). Interestingly, versican by microRNAs (miRNA). miR-199a* function as a guide molecule in post-transcriptional gene silencing by binding to region (3¢-UTR) of versican the 3¢-untranslational mRNA, leading to translational repression. miR-199a* is considered to be an onco-suppressor, targeting mole- cules critically involved in the promotion of tumor growth and is often downregulated in malignancies [39,40]. Several companies have developed miRNA- based therapeutics and a strategy that increases the natural dose of miR-199a*, by introducing a short double-stranded synthetic RNA that is loaded into RNA-induced silencing complex or by utilizing expres- the hairpin pre-miRNA in a viral vector sion of

Aggrecan is a large molecule found mainly in cartilage and brain, almost exclusively in the form of aggregates with HA (Fig. 1). It is a main matrix organizer also involved in the regulation of cartilage development, growth and homeostasis. Several studies describe its involvement in malignancies. Aggrecan is markedly reduced and degraded at speciﬁc sites during cartilage destruction in the progression of laryngeal squamous cell cancer [47,48] (Table 1).

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Brevican: roles in gliomas

Biological roles of SLRPs / decorin

Formation and spacing of collagen fibrils

Collagen I

SLRPs

Decorin

EGFR

c-Met

P

P c-Cbl

role in glioma progression.

In addition,

Induction of p21

Signaling modulation proteasome degradation

Brevican and neurocan, found in the central nervous system, affect neuronal attachment and neurite out- growth (Fig. 1). Upregulation and proteolytic cleavage of brevican increase the aggressiveness of glial tumors signiﬁcantly [49] and enhance cell adhesion and motil- ity. Brevican promotes EGFR activaton, increases the expression of cell-adhesion molecules and promotes the secretion of ﬁbronectin and accumulation of ﬁbro- nectin microﬁbrils on the cell surface [50]. The expres- sion of a novel tumor-speciﬁc isoform of brevican that is localized on the cell membrane has been found in all high-grade gliomas and is suggested to play a signiﬁ- cant the absence of brevican from benign gliomas prompts its use as a diagnostic marker to distinguish primary brain tumors of similar histology, but different pathologic course [51]. Inhibition of the expression of the tumor- speciﬁc isoform of brevican and inhibition of brevican cleavage may be a potential pharmacological target for the treatment of brain tumors (Table 1).

Inhibition of tumor cell growth and migration

Endocytosis degradation

Small leucine-rich PGs

Fig. 3. SLRPs have a curved molecular architecture and bind to col- lagen ﬁbrils through the core protein participating in the formation and spacing of the ﬁbrils. Decorin binds to the EGFR and evokes a unique signaling cascade that causes EGFR dimerization, caveolin- mediated internalization and lysosomal degradation. EGFR is phos- phorylated upon decorin binding, and induces the expression of p21 leading to growth arrest. Decorin binds also to the c-Met induc- ing transient activation of the receptor recruitment of c-Cbl and rapid intracellular degradation of the receptor by the proteasome. Upon decorin binding to c-Met, intracellular levels of b-catenin are suppressed inhibiting tumor cell growth and migration.

SLRPs are characterized by a protein core with leu- cine-rich repeats, the presence of N-terminal cysteine clusters and C-terminal ‘ear repeats’ (classes I–III), and at least one GAG side chain [52]. The family of SLRPs is now divided into ﬁve classes (see the minire- view by Iozzo & Schaefer [53]). Most of these SLRPs have a consensus sequence for modiﬁcation with GAGs, but some exist as glycoproteins in the tissues. SLRPs are important regulators of various biological processes because leucine-rich repeats are particularly relevant for protein–protein interactions [52]. SLRPs have a curved molecular architecture and bind to col- lagen ﬁbrils through a core protein, participating in the formation and spacing of the ﬁbrils (Fig. 1). Most studies on the role of SLRPs in malignancy have focused on decorin and lumican, which are typically located around tumor microenvironment.

Decorin signaling in cancer

Decorin represents a powerful tumor cell growth and migration inhibitor by modulating both tumor stroma deposition and cell-signaling pathways. In addition, genetic evidence suggests that a lack of decorin is ‘per- missive’ for tumor development [54]. Growth arrest of lines has been associated with various tumor cell induced expression of inhibitors of cyclin-dependent [55,56]. Notably, decorin binds kinase p21 (Fig. 3)

directly to EGFR and downregulates its activity as well as the activity of other members of the ErbB fam- ily (Fig. 3) [55,57]. These receptors are overexpressed and ⁄ or mutated in many cancers driving tumor pro- gression [58]. Decorin can compete with EGF for receptor binding on the cell surface of tumor cells. After binding, the receptor dimerizes and is subse- quently internalized and degraded in the lysosomes via caveolin-mediated internalization (Fig. 3) [59]. Decorin inhibits tumor cell proliferation by evoking a signaling cascade that is different from the one evoked by EGF, possibly by inducing a different EGFR conformation and selectively activating phosphotyrosines in the receptor autophosphorylation domain. Decorin also suppresses the activity of ErbB2 and ErbB4 receptors via degradation (Fig. 3) [57,60]. The only exception to

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recruitment of

further investigation. Decorin expression in cancer can also be altered by transcriptional, post-transcriptional and post-translational modiﬁcations. Notably, decorin production is re-established following treatment with a proteasome inhibitor, suggesting that ovarian cancer cells have developed a mechanism for the rapid degra- dation of decorin [67]. Potentially, epigenetic control, including hypermethylation of the promoter region, might play a role in silencing the decorin gene. In the tumor stroma, by contrast, hypomethylation of the decorin promoter has been previously demonstrated [1]. In addition, tumor cells can synthesize soluble factors that repress decorin expression by stromal cells [70].

this model reported to date has been in MG-63 human osteosarcoma cells, in which the decorin-expressing tumor cells show overexpression paired to constitutive activation of the EGFR favoring cell migration [61]. Decorin also interacts with Met, the receptor for hepa- tocyte growth factor, and induces transient receptor activation, the E3 ubiquitin ligase c-Cbl, and rapid intracellular degradation of the recep- tor (Fig. 3). Decorin suppresses intracellular levels of b-catenin, one of the key downstream effectors of Met, and inhibits cell migration and growth (Fig. 3). Thus, by antagonistically targeting multiple tyrosine kinase receptors, decorin contributes to the reduction in pri- mary tumor growth and metastastic spreading [62].

Decorin as potential anticancer agent

Decorin as a marker for prognosis and aggressiveness

Decorin expression is altered in various types of cancer (Table 1). Therefore, decorin has been taken into con- sideration as a possible prognostic marker in cancer patients. It is important to note that decorin expres- sion levels are directly proportional to the amount of tumor stroma. There are very few studies that analyze the prognostic signiﬁcance of decorin expression levels in terms of patient survival. High expression of decorin in patients with advanced ovarian cancer was associ- ated with a poor response to treatment and a higher incidence of relapse for those patients that initially responded [63]. Reduced amounts of decorin were associated with poor prognosis in node-negative inva- sive breast cancer [64] and some types of soft tissue tumors [65]. Low decorin levels in liposarcomas and malignant peripheral nerve sheath tumors are also associated with lower disease-free and survival rates (Table 1). Decorin is also deposited in the tumor stroma in nonpalpable breast carcinomas as versican. The accumulation of decorin is more pronounced than that of versican and is also associated with cancer-sug- gesting mammogram ﬁndings (personal data).

[66].

In normal tissues, the ratio of CS and DS side chains associated with decorin is balanced. In tumor tissue, such as colon, ovarian, gastric and pancreatic the CS chains become predominant carcinoma, [66–69]. DS is chemically more complex and requires additional enzymes to be synthesized, compared with CS. Therefore, it is reasonable to think that the syn- thesis of the chemically simpler CS is favored in tumor tissue. In line with this concept, CS side chains have been proposed as being more permissive to cell migra- tion favoring tumor aggressiveness is not surprising that, tumor cells, with very few exceptions, do not express decorin, but the mechanism by which decorin expression is switched off is unclear and needs

Decorin will attract more interest in future as an anti- cancer therapeutic. Considering that chemotherapy is still the leading therapy for cancer patients and that decorin could be administered in concomitance with various compounds, it is relevant to understand their interaction. Decorin shows a synergistic biological effect with carboplatin in inhibiting ovarian cancer cell growth [71], whereas it antagonizes carboplatin and gemcitabine effects against pancreatic cancer cells [72]. Because of a lack of data and somewhat contrasting results, this area deﬁnitely needs more investigation. Considerable effort has been recently applied to prove that decorin can be an antitumor therapeutic in vivo. the Adenoviral-mediated delivery of decorin slows growth of lung, squamous and colon carcinoma tumor xenografts in immunocompromised mice [73], retards mammary adenocarcinoma growth and prevents meta- static spreading to the lungs reducing ErbB2 receptor levels [66]. Ectopic expression of decorin in a rat glioma model prolongs the survival of the animals and the size of the tumor is directly proportional to how early and how much decorin is expressed [74]. Admin- istration of decorin protein core showed that it speciﬁ- cally localizes within the tumor, antagonizes EGFR activity and induces apoptosis in the A431 squamous carcinoma model [75]. The outcome is primary tumor growth inhibition because of the slower growth rate combined with apoptosis and impaired tumor metabo- lism. Decorin injected systemically can reduce breast tumor growth and metabolism and halt metastatic spread to the lungs [76]. The ﬁnding that ectopic expression of decorin can revert the malignant pheno- type in several cell lines of various histogenetic back- grounds [56] and can antagonize primary tumor growth and metastases in vivo further raises a hope for the postulated clinical application of decorin and related molecules. Decorin might be utilized in the

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near future as an adjunct ‘protein therapeutic’ for solid tumors in which receptor tyrosine kinases play a key in vivo studies with various tumor role. Additional models are desirable. Furthermore, studies to elucidate the functional interaction between decorin and existing anticancer chemotherapeutics to evaluate in order potential limitations are needed (Table 1).

Lumican: a promising agent in the cancer ﬁght

inhibit melanoma cell migration [81]. It will be interest- ing in future to test the effects of recombinant lumican on tumor growth and metastasis. Additional studies carried out with different cancer types are required to dissect the mechanism of action of lumican. However, it appears that lumican targeting is promising and more speciﬁc information about its mode of action is needed. SLRPs or peptides derived therefrom could be applied in the ﬁght against cancer, because they repre- sent a class of natural inhibitors of cancer growth (Table 1).

anchorage-independent

Basement membrane PGs

[78] and its

Lumican is substituted with keratan sulfate side chains, and inhibits cell proliferation by inducing p21 expres- cell sion and antagonizes growth as well as cell migration [77]. Cleavage of lumi- can by the membrane-type matrix metalloprotease-1 abrogates lumican-evoked suppression of colony for- mation [77]. Interestingly, and on the whole contrary to decorin, lumican is expressed by some tumor cell lines inhibition promotes cancer cell growth in some cases. Transfection of B16F1 mouse melanoma cells to express lumican or treatment with recombinant protein induces impaired anchorage-inde- pendent growth and the capacity to invade the ECM [79]. Lumican has been reported to be more abundant than decorin in human breast carcinomas [80], even though the clinical relevance of this observation has not yet been explained. More importantly, lumican expression has been proposed as a prognostic factor in lymph node-negative breast cancer [64]. A recent study tested different recombinant and synthetic peptides and found an active site in the leucine-rich repeat 9 domain of the lumican core protein, which is able to

Basement membranes are thin layers of specialized ECM underlying epithelial and endothelial layers; a located in basement speciﬁc class of matrix PGs membranes with particular structural and functional characteristics. Basement membranes are elongated molecules with a collage of domains that share struc- tural and functional homology with numerous ECM proteins, growth factors and surface receptors. This class involves three main, well-characterized members: perlecan, collagen type XVIII and agrin, which are almost universally decorated with HS side chains (Fig. 4) [82]. Their genes are highly conserved and carry disparate biological functions for the mainte- nance of basement membrane homeostasis, modulation of growth factor activity and angiogenesis. Basement membrane heparin sulfate proteoglycans (HSPGs) have a dual function as pro- and anti-angiogenic factors participating in cancer growth and angiogenesis [82].

Endorepellin

Perlecan

SEA

GAG chains

Laminin-EGF-like domain repeats

Laminin domain IV

LDL-receptor class A repeat

EGF-like repeats

Ig-like repeats

Laminin G domain

Agrin

Follistatin-like repeats

Basement membrane PGs

Ser / Thr-rich domains

N-terminal agrin

Endostatin

C-terminal domain

N-terminal domain

Collagen XVIII

Non-collagenous sequences

Interrupted triple helical domain

Fig. 4. Schematic representation showing the structural domains of the basement membrane PGs perlecan, agrin and collagen XVIII.

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They can stimulate angiogenic signaling by sequester- ing, protecting and concentrating HS-binding growth factors, such as ﬁbroblast growth factor-2 (FGF-2), VEGF and PDGF, through which the HSPG growth factor complex may be presented in a ‘biologically active’ form to the cognate receptors (Fig. 5). By con- trast, they contain powerful angiostatic fragments, such as endostatin and endorepellin at their C-termini that are released by proteolytic processing and can act in a paracrine function on sprouting endothelial cells, either locally or distantly (Fig. 5).

Perlecan: a critical regulator of growth factor- mediated signaling and angiogenesis

angiogenesis

tumor

and

response, as

Perlecan binds FGF-2 via HS, promotes receptor acti- vation and ultimately downstream signaling, which supports mitogenesis and angiogenesis (Fig. 5). The lack of HS in perlecan inhibits wound healing and FGF-2-induced growth [82,83]. Targeted knockdown of perlecan reduced the revealed by decreased growth factor tumor growth and angiogenesis [82,84]. In hepatoblas- toma xenografts treated with anti-vascular endothelial

growth factor receptor (VEGFR) therapy, vessel recov- ery over time was associated with an increase in perlec- an and heparanase expression around tumor vessels [85], suggesting a synergistic role of heparanase [86] in the liberation of HS-bound and ⁄ or proteases [87] VEGF and subsequent VEGFR2 activation (Fig. 5). The C-terminal domain of perlecan, called endorepel- lin, blocks endothelial cell migration and capillary morphogenesis both in vitro and in vivo [82]. Endore- pellin interacts speciﬁcally with the a2b1 integrin [88], and stimulation with endorepellin induces the interac- tion and phosphorylation of Src homology-2 protein phosphatase-1 with integrin a2 in a dynamic fashion in endothelial cells [89], triggering a signaling cascade that leads to disruption of the actin cytoskeleton and thus to cytostasis (Fig. 5) [88]. Systemic delivery of human recombinant endorepellin to tumor xenograft- bearing mice causes a marked suppression of tumor growth and metabolic rate mediated by a sustained downregulation of the tumor angiogenic network [90]. The distal laminin-like globular domain (LG3) pos- sesses most of the biological activity [82,88] and can be released from the parent molecule by bone morphoge- [91]. netic protein-1 ⁄ Tolloid-like metalloproteinases

Biological roles of basement membrane PGs

Perlecan

GF

Agrin

Protection of growth factors chemotactic gradient

Tumorigenesis angiogenesis

GF

Collagen XVIII

GF

Endostatin

Endorepellin

Glypicans

VEGFR

Growth factor receptor

GF

α β

GF

G F G F

Integrins

P

Growth factor receptor signaling

Disruption of actin cytoskeleton down-regulation of VEGF signaling synthesis of thrombospondin

Inhibition of angiogenesis

Tumor / endothelial cell proliferation and motility

Fig. 5. PGs of the basement membrane bind HS-binding growth factors and stimulate angiogenesis and tumorigenesis by sequestering, pro- tecting and concentrating growth factors. HSPG–growth factor complexes are presented in a ‘biologically active’ form to the cognate recep- tors promoting their signaling. By contrast, they contain angiostatic fragments, such as endostatin and endorepellin, which are released by proteolytic cleavage. Endorepellin interacts with a2b1 integrin inducing the interaction and phosphorylation of src homology-2 protein phos- phatase-1 with integrin a2, triggering a signaling cascade that leads to disruption of the actin cytoskeleton and thus to cytostasis. Endostatin binds to integrins (a5b1, avb3, avb5), glypican and VEGFR2 affecting several key components of VEGF signaling cascade, stimulating synthe- sis of thrombospondin (a powerful angiostatic protein) and suppressing c-myc. Endostatin induces reprogramming of the gene expression and disrupts endothelial cell migration.

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macroencapsulation of engineered cells that produce endostatin may be innovate therapeutic strategy for treatment of malignancies (Table 1).

Agrin: roles in tumor angiogenesis

layer of control

LG3 has been detected in pathological conditions including pancreatic, colon and breast cancer [82]. It has been proposed that endorepellin ⁄ LG3 is liberated via partial proteolysis during tissue remodeling and cancer growth, thereby representing an additional layer of control for angiogenesis [82]. One possibility is that tumor growth might be enhanced in vivo by a lack of circulating LG3. In line with this, circulating LG3 lev- els are reduced in patients with breast cancer, suggest- ing that reduced titers might be a useful biomarker for cancer progression and invasion [92]. The fragment in circulation might be continuously released to add an for angiogenesis during additional cancer progression. Utilization of the antiangiogenic fragment as either a protein- or peptide-based pharma- cological agent might represent a novel therapeutic rationale, especially when provided in combination with other tumor-suppressive compounds (Table 1).

Collagen XVIII–endostatin: innovative angiogenic regulators

Agrin is strongly expressed around blood vessels [82]. Agrin can be considered to be a marker of tumor angiogenesis in the liver because it is markedly depos- ited in proliferating bile ductules, in newly formed sep- tal vessels in hepatic cirrhosis and in the angiogenic network of malignant hepatocellular carcinomas (HC) [99]. Agrin overexpression in newly formed blood ves- sels is also present in cholangiocarcinoma [100]. The role of agrin in tumor angiogenesis is not yet estab- lished, but initial studies suggest that agrin’s effect on tumor angiogenesis is likely context dependent. It seems that the expression of agrin may be protective against disorganized angiogenesis in glioblastomas, whereas in hepatic malignancies it seems to support tumor angiogenesis, at least in the initial stages of tumor development. There remains very limited knowl- edge of whether C-terminal endorepellin-like fragment of agrin signals through the integrin receptors. Further research on this area will improve our knowledge for possible targeting of agrin in tumors (Table 1).

Cell-surface PGs

type I

(Fig. 6). Syndecans

The cell-surface PGs include two major subfamilies: the syndecans, with four members cloned in mammals, which are transmembrane proteins mostly substituted with HS chains; and glypicans linked with a glycosyl-phosphatidylinositol anchor to the cell mem- brane, with six members cloned in mammals substi- tuted with HS chains and glypicans are generally considered to act as coreceptors for heparin-binding mitogenic growth factors [2].

Glypicans inﬂuence tumor development and progression

Collagen XVIII has been suggested to play a negative regulatory, but not essential, role in angiogenesis in certain contexts. Collagen type XVIII harbors the C-terminal antiangiogenic fragment, endostatin, pro- teolytically derived from the C-terminus of collagen type XVIII [82,93]. The expression of collagen XVIII and endostatin has been studied in ovarian, hepato- cellular, pancreatic and oral squamous cell carcinoma and found to vary among different cancer types [94]. Endostatin is a potent antiangiogenic molecule reduc- ing tumor growth, choroidal neovascularization and wound healing [82]. High levels of circulating endosta- tin reduce tumor burden, block the formation of pul- monary metastases [95] and, notably, induce a total gene expression reprogramming [96], which ultimately disrupts endothelial cell migration (Fig. 5) [97]. En- dostatin has been linked to cell-surface receptors including various integrins, glypican and VEGFR2. Speciﬁcally, endostatin downregulates several key com- ponents of the VEGF signaling cascade and, at the same time, stimulates the synthesis of thrombospondin, a powerful angiostatic protein and suppresses c-myc (Fig. 5) [82,96]. Endostatin has been studied in phase I and II clinical trials for patients with metastatic cancer and has shown low efﬁcacy, however, a new more sta- ble version of endostatin has re-entered the clinic and is now used in certain countries for the treatment of lung and gastric cancer [94]. Recently, an immunoiso- lation device that contains endostatin-expressing cells was used effectively for the treatment of melanoma and Ehrlich tumors in mice [98]. This suggests that the

Glypicans show cell-type and developmental-stage-spe- ciﬁc expression. They are involved in fundamental bio- logical processes such as cell–ECM interactions and the control of cellular division, differentiation and morphogenesis [101]. At the level of signaling, they are involved in the regulation of pathways including Wnt, FGF, Hedgehog (Hh), bone morphogenic protein, Slit and insulin-like [101]. Therefore, growth factor depending on the biological context, glypicans can either stimulate or inhibit signaling activity. Notably, the HS chains are essential for the glypican-induced

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Intracellular and cell-surface PGs

Syndecans

Nucleus

Serglycin

GAG chains

Glypicans

its

endocytosis

GPI-anchor

Fig. 6. Localization and classiﬁcation of cell-surface associated and intracellular PGs. The cell-surface PGs encompass mainly the trans- membrane PGs syndecans and the glycosyl-phosphatidylinositol- anchored glypicans. Serglycin is the only characterized intracellular PG.

stimulation of FGF activity, and partially required for the regulatory activity of glypicans in Hh, Wnt and bone morphogenetic protein signaling [101–104].

Glypicans inﬂuence tumor development and progres- sion, and their expression is abnormal in various human tumors (Table 1). For example, glypican-1 is upregu- lated in breast and pancreatic cancer, and it has been postulated that this aberrant expression of glypican-1 may play a key role in promoting growth factor signal- ing in cancer cells [105,106]. By contrast, glypican-3 gene is mutated in patients with Simpson–Golabi– Behmel syndrome, deregulating the balance between cell proliferation and apoptosis [107]. The loss of glypi- can-3 induces overgrowth observed in this syndrome. Patients with Simpson–Golabi–Behmel syndrome are

increased risk of embryonic tumors. Glypican-3 at inhibits the signaling of Hh by competing with Patched, the Hh receptor, for Hh binding [102]. The binding of Hh to the receptor Patched triggers the sig- naling pathway by blocking the inhibitory effect of Patched on Smoothened. Glypican-3 competes with Patched for Hh binding allowing to ligand-free Patched to inhibit Smoothened, reducing signaling and cell growth (Fig. 7). The binding of Hh to glypican-3 triggers and degradation further strengthens the negative regulatory role of glypican-3 in Hh signaling (Fig. 7). Glypican-3 plays a negative role in cell proliferation and induces apoptosis in mesothelioma and breast cancer that is only dependent on the protein core [108]. Glypican-3 expression is downregulated in tumors with different histogenetic backgrounds as a result of hypermethylation of the glypican promoter, and its expression can be restored by treatment with demethylation agents [109–112]. Hh signaling pathway is hyperactivated in a proportion of these cancers [113,114] and it is therefore possible that the loss of glypican-3 may contribute to cancer pro- gression by inducing the activation of Hh signaling similarly to Simpson–Golabi–Behmel syndrome. By contrast, upregulation of glypican-3 is observed in embryonic tumors, such as neuroblastoma and Wilm’s tumor [115]. In tumors originating from tissues that express glypican-3 only in the embryo, such as HC and melanoma, its expression tends to reappear with transformation [101]. The canonical Wnt malignant pathway plays a key role in most HC and overexpres- sion of glypican-3 promotes tumor growth-promoting Wnt signaling. The binding of Wnt to glypican-3 facili- tates and ⁄ or stabilizes the interaction of Wnt with the

Biological roles of glypican 3 and serglycin

Protection, transport, activation and delivery of bioactive molecules

Glypican 3

Hh

Wnt Wnt

Hh Hh

Frizzled

Patched

Smoothened

Patched

X

Hh

Serglycin

Increased signal

Reduced signal

Increased signal

Endocytosis degradation

Bioactive molecules

Regulation of tumor growth

Fig. 7. The binding of Wnt to glypican-3 facilitates and ⁄ or stabilizes the interaction of Wnt and Frizzled with the consequent increament on signaling. The binding of Hh to the receptor Patched triggers the signal- ing pathway by blocking the inhibitory effect of Patched on Smoothened. Glypican-3 competes with Patched for Hh binding allowing ligand-free Patched to inhibit Smoothened, reducing signaling and tumor growth. The binding of Hh to glypican-3 triggers endocytosis and degradation of the complex. Serglycin interacts with and stores bioactive molecules inside storage granules and secretory vesicles. Upon secretion, serglycins modulate their activities through protection, transport, activation and delivery of the bioactive molecules.

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receptor Frizzled with the consequent increment on signaling (Fig. 7). HC cells forced to overexpress a sol- uble form of glypican-3 showed lower tumorogenicity because soluble glypican-3 blocked the activity of sev- eral pro-tumorigenic growth factors. Glypican-3 may promote local cancer growth in some cancer tissues, whereas it inhibits tissue invasion and metastasis in others.

eradication by T cells of

accelerate

shedding

It is suggested that cell-surface syndecans promote pro- angiogenic signaling by binding FGF-2 and VEGF and presenting them to their high-afﬁnity receptors and also by protecting them from inactivation (Fig. 8) [127,128]. Notably, syndecan-1 also mediates cell adhe- sion in cooperation with integrins by activation of actin and fascin bundling (Fig. 8) [119,124]. By con- trast, syndecan-1 is downregulated in various malig- nancies and a possible inhibitory function is suggested [119,129–131]. Syndecan-1 may serve as a prognostic marker in a cancer-type-speciﬁc manner (Table 1). The effects of syndecan-1 on tumor progression and prog- nosis probably also depend upon whether syndecan-1 is tumor cell derived or synthesized by the host stroma, because its expression by reactive tumor stromal cells promotes tumor progression [123,125,126,132–135]. Soluble syndecan-1 ectodomain is present in the serum of various malignancies. A variety of proangiogen- ic ⁄ protumor growth factors and enzymes that are up- regulated in solid tumors in vivo (e.g. FGF-2 and in vitro MMPs) syndecan-1 [136,137], presumably by promoting cleavage of the accelerates ectodomain [138]. Soluble

syndecan-1

Biological roles of syndecans

Soluble syndecans

Tumorigenesis angiogenesis

Glypican-3 is a novel tumor marker for early-stage melanoma [116] and early-stage HC [117]. It is not known, however, whether its elevated serum levels are important in tumor progression or are simply a reﬂec- tion of aggressive tumors. The high levels of shed glyp- ican may arise from increased expression of the PG or increased activity of tumor proteases. In this light, glypican-3 may be a candidate antigen for cancer strongly immunotherapy in HC (Table 1). is is highly immunogenic expressed exclusively in HC, and stimulates tumors expressing glypican-3 in mice and markedly inhibited growth of an established tumor. Glypican-3-derived peptide-pulsed vaccination is a novel strategy to pre- vent HC and melanoma in patients and needs to be further developed as an anticancer therapy [116]. Glyp- ican-1 on target cells is recognized by some natural cytotoxicity receptors of natural killer cells and the suppression of glypican-1 in pancreatic cancer led to lower cytotoxic effects of natural killer cells [118] (Table 1).

Protection of proteases and growth factors Presentation to receptors Chemotactic gradient Competition with cell surface syndecans

GF GF

GF

Fibronectin

Syndecans

Growth factor receptor

Syndecans: multiple roles in cancer progression and strategies for their targeting

Collagen

α β

GF

Integrins

P

Membrane ruffling and stress fiber formation

Growth factor receptor signaling

through which

regulate

events

they

Tumor / endothelial cell adhesion and migration

Tumor / endothelial cell proliferation and motility

Syndecan-1 is present at early stages during develop- ment and on epithelial and cancer cells in adults. Synd- ecan-2 is distributed in mesenchymal tissues, liver and neuronal cells. Syndecan-3 is mainly associated with neural tissues, whereas syndecan-4 is ubiquitously dis- tributed [2]. Syndecans are involved in complex signal- ing cell proliferation, differentiation, adhesion and migration [119]. For the multiple roles of syndecan shedding see the accompanying minireview by Manon-Jensen et al. [120].

Fig. 8. Syndecans interact with matrix molecules in cooperation with integrins, inducing membrane rufﬂing and stress ﬁber forma- tion, promoting cell adhesion and migration. Syndecans bind to growth factors presenting them to their high-afﬁnity receptors, acti- vating signaling pathways that promote cell proliferation and motil- ity. Soluble syndecans interact with growth factors protecting them from degradation and creating a promigratory chemotactic gradient. Soluble syndecans present growth factors to their high-afﬁnity receptors, activating signaling pathways that promote cell prolifera- tion and motility. Soluble syndecans bind to matrix molecules, com- petitively inhibiting cell-surface syndecans, and thereby stimulating tumor and endothelial cell migration.

Syndecan-1 is expressed in various human cancers and correlates with tumor recurrence in human pros- it is upregulated in tate cancer [121]. Furthermore, human breast cancer and correlates with poor progno- sis [122]. Syndecan-1 expression by ﬁbroblasts in the tumor-associated stroma appears to be necessary for breast carcinogenesis [123] and may promote tumori- genesis by regulating tumor cell spreading and adhe- sion [124], proliferation and angiogenesis [125,126].

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factors,

tumor growth in vivo and increased cell invasion through collagen in vitro [139]. Soluble syndecan-1 may activate proangiogenic ⁄ protumor like FGF-2 [140], also bind to mitogens, effectively creating a chemotactic gradient [141] and ⁄ or interact with pro- teases protecting them from contact with endogenous inhibitors (Fig. 8) [119].

are

tumors

these

endothelial

signaling, including protein kinase Ca and focal adhe- sion kinase activation, to promote focal adhesion for- mation (Fig. 8) [148]. FGF-2 treatment of melanoma cells resulted in reduction in the expression of syndec- an-4, a decrease in cell attachment on ﬁbronectin and promoted cell migration [149]. Syndecan-4 overexpress- ing cells form larger and more dense focal adhesions, correlated with stronger attachment and decreased cell migration [150], whereas lack of syndecan-4 engage- ment promotes an amotile ﬁbroblast phenotype in which focal adhesion kinase and Rho signaling are downregulated and ﬁlopodia extended [151] (Table 1). Syndecan-4 is highly expressed in less- aggressive seminomatous testicular tumors. Reduced levels are detected in more-aggressive non-seminoma- tous germ cell tumors and it correlates with the meta- (personal data). static potential of Interestingly, syndecan-4 is found also in the tumor stroma in some testicular tumors and the presence of stromal-associated syndecan-4 correlates with increased neovascularization. These results indicate that a direc- ted homeostasis in syndecan-4 levels supports the opti- mal migration of tumor cells and its expression by stromal cells may facilitate cancer cell growth and angiogenesis, similarly to syndecan-1.

soluble

assembly by

competing

for

Overexpression of syndecan-2 has been found in numerous malignancies, such as Lewis lung carcinoma, ovarian, osteosarcoma, brain tumors, mesothelioma and colon cancer [119,142–144] (Table 1). This PG promotes tumorigenesis in colorectal cancer and Lewis lung carcinoma by regulating cell adhesion, spreading and cytosketetal organization [142,144]. Recently, we found that expression of syndecan-2 in breast cancer cells is regulated by estradiol through the action of estrogen receptor alpha [145]. Syndecan-2 is highly expressed in the microvasculature of mouse glioma tumors and promotes angiogenesis, at least in part, by modulation of endothelial cell adhesion [143]. Exoge- nous addition of EGF, FGF-2, VEGF, MMP-2 and cells MMP-9 to brain microvascular increases syndecan-2 ectodomain shedding [143]. The released ectodomain may act on tumor cells and ⁄ or the endothelium to modulate tumorigenesis. Soluble syndecan-2 ectodomain causes signiﬁcant increase in endothelial cell membrane protrusion, migration, capil- lary tube formation and cell–cell interactions most probably because of competitive inhibition of cell-sur- face syndecan- 2 (Fig. 8) [119,143]. Colorectal carci- noma cells demonstrate reduced cell adhesion in the syndecan-2 ectodomain [143], presence of whereas overexpression of full-length syndecan-2 in lung carcinoma cells induces stress ﬁber formation and cytoskeletal structures indicative of strong adhesion; cells also become nonmotile [142]. Cell-surface syndec- an-2 is also needed for the correct organization of lam- inin and ﬁbronectin, and its reduced interaction with matrix ligands because of the presence of soluble ecto- domain would be expected to decrease basement mem- brane laminin and ﬁbronectin binding [146]. Syndecan-2 ectodomain may exert its proangiogenic effect by binding to growth fac- tors and cytokines [119], effectively creating a chemo- tactic gradient similar to that produced by soluble syndecan-1 (Fig. 8) [141].

Syndecan-4 is a focal adhesion component

in a range of cell types and mediates breast cancer cell adhesion and spreading [119]. It also forms a complex with the proangiogenic molecule, FGF-2, and its receptor, ﬁbroblast growth factor receptor (FGFR-1) promoting FGF signaling (Fig. 8) [147]. The attach- ment of syndecan-4 to ﬁbronectin initiates intracellular

The antitumor effect of murine ⁄ human chimeric syndecan-1-speciﬁc monoclonal antibody nBT062 con- jugated with highly cytotoxic maytansinoid derivatives against multiple myeloma cells in vitro and in vivo was examined. Treatment signiﬁcantly inhibited multiple myeloma tumor growth in vivo and prolonged host survival in both the xenograft mouse models of human multiple myeloma and the SCID-hu mouse model. These results provide a preclinical framework support- ing the evaluation of nBT062-maytansinoid derivatives in clinical trials to improve patient outcome in multiple myeloma [152] (Table 1). Another strategy for syndec- an targeting in the treatment of malignancies is the inhibition of heparanase an enzyme that degrades HS (see the accompanying minireview by Barash et al. [153]). 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 lead- ing to enhanced phosphorylation of selected protein kinases and increased gene transcription associated with aggressive tumor progression. This function is mediated by a novel protein domain localized at the heparanase C-terminus. Heparanase is also an impor- tant regulator of syndecan clustering, shedding and mitogen binding. Recent data indicate that modiﬁed glycol-split heparin, as well as other heparanase inhibi- tors, can profoundly inhibit the progression of tumor

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4 Wight TN (2002) Versican: a versatile extracellular

xenografts produced by myeloma and carcinoma cells, thus moving antiheparanase therapy closer to reality [154] (Table 1).

Intracellular PGs

matrix proteoglycan in cell biology. Curr Opin Cell Biol 14, 617–623.

5 Wight TN & Merrilees MJ (2004) Proteoglycans in ath- erosclerosis and restenosis: key roles for versican. Circ Res 94, 1158–1167. 6 Theocharis AD (2008) Versican in health and disease. Connect Tissue Res 49, 230–234.

(e.g. histamine,

7 Wu YJ, Lapierre D, Wu J, Yee AJ & Yang BB (2005) The interaction of versican with its binding partners. Cell Res 15, 483–494. 8 Kim S, Takahashi H, Lin WW, Descargues P,

Grivennikov S, Kim Y, Luo JL & Karin M (2009) Carcinoma-produced factors activate myeloid cells through TLR2 to stimulate metastasis. Nature 457, 102–106.

9 Sakko AJ, Ricciardelli C, Mayne K, Tilley WD, Le- Baron RG & Horsfall DJ (2001) Versican accumula- tion in human prostatic ﬁbroblast cultures is enhanced by prostate cancer cell-derived transforming growth factor beta1. Cancer Res 61, 926–930. 10 Ricciardelli C, Brooks JH, Suwiwat S, Sakko AJ,

Serglycin is the only characterized intracellular PG found in hematopoietic and endothelial cells (Fig. 6). It has important functions related to the formation of sev- eral types of storage granules. Serglycin interacts with several bioactive molecules tumor necrosis factor a and various proteases) and is impor- tant for the retention of these key inﬂammatory media- tors inside storage granules and secretory vesicles. Serglycin can further modulate the activities of partner molecules in different ways after secretion from acti- vated immune cells, through protection, transport, acti- vation and interactions with substrates or target cells (Fig. 7) [155]. Few reports involve serglycin in malig- nancies. Niemann et al. [156] demonstrated the expres- sion of serglycin in various hematologic malignancies. Serglycin expression was found to distinguish acute myeloid leukemia from acute lymphoblastic leukemia. It was found to be a selective marker for immature myeloid cells, distinguishing acute myeloid leukemia from Philadelphia chromosome-negative chronic myelo- proliferative disorders. The expression and constitutive secretion of serglycin has also been reported in multiple myeloma, where the released serglycin inhibits bone mineralization [157]. The role of serglycin in hemato- logical malignancies remains unkown and further studies will improve our knowledge of its involvement and potential pharmacological targeting (Table 1).

Mayne K, Raymond WA, Seshadri R, LeBaron RG & Horsfall DJ (2002) Regulation of stromal versican expression by breast cancer cells and importance to relapse-free survival in patients with node-negative pri- mary breast cancer. Clin Cancer Res 8, 1054–1060. 11 Nikitovic D, Zaﬁropoulos A, Katonis P, Tsatsakis A, Theocharis AD, Karamanos NK & Tzanakakis GN (2006) Transforming growth factor-beta as a key mole- cule triggering the expression of versican isoforms v0 and v1, hyaluronan synthase-2 and synthesis of hyal- uronan in malignant osteosarcoma cells. IUBMB Life 58, 47–53. 12 Arslan F, Bosserhoff AK, Nickl-Jockschat T, Doerfelt

Acknowledgement

We thank Prof. R.V. Iozzo for critical reading and valuable advice.

A, Bogdahn U & Hau P (2007) The role of versican iso- forms V0 ⁄ V1 in glioma migration mediated by trans- forming growth factor-beta2. Br J Cancer 96, 1560– 1568. 13 Serra M, Miquel L, Domenzain C, Docampo MJ,

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