Mammalian heparanase: involvement in cancer metastasis, angiogenesis and normal development

seminars in CANCER BIOLOGY, Vol. 12, 2002: pp. 121–129 doi:10.1006/scbi.2001.0420, available online at http://www.idealibrary.com on

Mammalian heparanase: involvement in cancer metastasis, angiogenesis and normal development Israel Vlodavsky∗ , Orit Goldshmidt, Eyal Zcharia, Ruth Atzmon, Zehava Rangini-Guatta, Michael Elkin, Tamar Peretz and Yael Friedmann

Basement membranes and heparan sulphate proteoglycans

Cleavage of heparan sulphate proteoglycans (HSPGs) affects the integrity and functional state of tissues and thereby fundamental normal and pathological phenomena involving cell migration and response to changes in the extracellular microenvironment. Heparanase, degrading heparan sulphate (HS) at specific intrachain sites, is synthesized as a latent ∼65 kDa protein that is processed at the N-terminus into a highly active ∼50 kDa form. The heparanase enzyme is preferentially expressed in human tumours and its overexpression in low-metastatic tumour cells confers a highly invasive phenotype in experimental animals. Heparanase also releases angiogenic factors and accessory fragments of HS from the tumour microenvironment and induces an angiogenic response in vivo. Heparanase may thus facilitate tumour cell invasion, vascularization and survival in a given microenvironment, all critical events in cancer progression. These observations, the anticancerous effect of heparanase-inhibiting molecules, and the unexpected identification of a single predominant functional heparanase suggest that the enzyme is a promising target for drug development.

The extracellular matrix (ECM) is a heterogeneous mixture of proteins and polysaccharides that surrounds cells and supports cellular organization into tissues and organs. Basement membranes (BM) are specialized ECM structures composed of characteristic macromolecules (i.e. collagen type IV, laminin, heparan sulphate proteoglycans) on which epithelial and endothelial cells migrate, proliferate and differentiate. 1 In carcinomas, BM often separate the tumour cells from the surrounding tissue and therefore present a main physical barrier limiting tumour invasion and metastatic spread. 1 Heparan sulphate proteoglycans (HSPGs) are ubiquitous macromolecules associated with the cell surface and ECM of a wide range of cells of vertebrate and invertebrate tissues. 2–5 The basic HSPG structure consists of a protein core to which several linear heparan sulphate (HS) chains are covalently O-linked. The polysaccharide chains are typically composed of repeating hexuronic and D-glucosamine disaccharide units that are modified at various positions by sulphation, epimerization and N-acetylation, yielding clusters of sulphated disaccharides separated by low or non-sulphated regions. 2–5 HS binds to and assembles ECM proteins, including fibronectin, laminins, and interstitial collagens, and plays important roles in cell–cell and cell–ECM interactions. 2–5 Moreover, the HS chains, unique in their ability to bind a multitude of proteins, ensure that a wide variety of bioactive molecules (i.e. heparin-binding growth factors, chemokines, lipoproteins, enzymes) bind to the cell surface and ECM and thereby function in the control of normal and pathological processes, among which are morphogenesis, tissue repair, inflammation, vascularization, and cancer metastasis 3–7 (Figure 1). Binding to HS can modulate the activity of the tethered molecule and/or protect the bound

Key words: metastasis / angiogenesis / heparanase / endoglycosidase / heparan sulphate proteoglycans / extracellular matrix c 2002 Elsevier Science Ltd. All rights reserved.

From the Department of Oncology, Hadassah-Hebrew University Hospital, Jerusalem 91120, Israel. *Corresponding author. Department of Oncology, Hadassah Hospital, POB 12000, Jerusalem 91120, Israel. E-mail: [email protected] c 2002 Elsevier Science Ltd. All rights reserved.

1044–579X / 02 / $ – see front matter

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Morphogens

Growth factors

HS Angiogenic factors Cytokines and chemokines

Heparanase Cleavage

Enzymes

Perlecan

ECM

Figure 1. Scheme showing the heparanase cleavage site (arrow) and the associated local release of bioactive molecules sequestered by HS in the ECM and tumour microenviornment.

proteins from proteolytic cleavage and inactivation. Apart from sequestration of bioactive molecules, transmembrane (syndecans) and phospholipidanchored (glypican) HSPGs mediate cell interactions with components of the microenvironment that control cell shape, adhesion, proliferation, survival and differentiation. 3,4 These species of HSPGs also have a co-receptor role in which the proteoglycan, in concert with the other cell surface molecules, comprises a functional receptor complex that binds the ligand and mediates its action. 3–7 Because of the important and multifaceted roles of HSPGs in cell physiology, their cleavage is likely to alter the integrity and functional state of tissues and provides a mechanism by which cells can respond rapidly to changes in the extracellular environment. In fact, enzymatic degradation of HS is involved in fundamental biological phenomena, ranging from pregnancy, morphogenesis and tissue remodelling to

inflammation, angiogenesis and cancer metastasis. This review focuses on the molecular properties, involvement in cancer progression and clinical significance of a mammalian endoglycosidase (heparanase), degrading HS.

Molecular and biochemical properties Mammalian HS-degrading enzymes, commonly referred to as heparanases, have been identified in a variety of normal and malignant cells and tissues, among which are cytotrophoblasts, endothelial cells (EC), keratinocytes, platelets, mast cells, neutrophils, macrophages, T and B lymphocytes, lymphoma, melanoma and carcinoma cells. 8–13 These enzymes cleave the glycosidic bond with a hydrolase mechanism and are thus distinct from bacterial eliminases, called heparinases and 122

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Figure 2.

Scheme of the human heparanase gene and protein.

heparitinase. Despite earlier reports on the existence of several distinct HS degrading endoglycosidases (heparanases), the cloning of a single gene by several groups 14–18 and subsequent biochemical characterization of the enzyme 19 suggest that mammalian cells express primarily a single dominant heparanase enzyme. The HS glycosaminoglycan chains are cleaved by heparanase at only a few sites, resulting in HS fragments of still appreciable size (10–20 sugar units). This result indicates that the enzyme recognizes a particular and quite rare HS structure. 20 A 2-O sulphate group on a hexuronic acid residue located two monosaccharide units away from the cleavage site appears essential for substrate recognition by heparanase 20 (Figure 1). In other studies, however, the presence of either Nor O-sulphates was not an absolute requirement for substrate cleavage. 21 The heparanase gene (∼50 kb) is located on human chromosome 4q21.3 and is linked to the genetic marker D4S400. 13,22 The gene is expressed as 5 and 1.7 kb mRNA species, generated by alternative splicing. The 5 kb form contains 14 exons and 13 introns, whereas in the short form the first and 14 exons have been spliced out. 22 The heparanase cDNA contains an open reading frame of 1629 bp that encodes for a 61.2 kDa polypeptide of 543 amino acids. The mature active 50 kDa enzyme, isolated

from cells and tissues, has its N-terminus 157 amino acids downstream from the initiation codon, 13–18 suggesting post-translational processing of the heparanase polypeptide (Figure 2). Processing and activation occur during incubation of the full-length 65 kDa recombinant enzyme with several normal and transformed cells, and to a lesser extent with their conditioned medium. 14 The nature of the cellular, apparently membrane bound, enzyme(s) involved in activation of the latent heparanase has not been characterized. Heparanase activity is readily obtained after transfection of mammalian cells with cDNAs encoding the entire heparanase precursor. 14–18 Attempts to express the truncated 50 kDa (Lys158 Ile543 ) protein failed, however, to yield active enzyme, suggesting that the region N-terminal to Lys158 plays a functional role in mediating expression and/or function of the protein. In fact, the active enzyme has been postulated to be a heterodimer of the 50 kDa subunit non-covalently associated with an 8 kDa peptide (Gln36 -Glu109 ), which arises from proteolytic processing of the pre-proheparanase protein 18 (Figure 2). The heparanase sequence contains a putative N-terminal signal peptide sequence (Met1 -Ala35 ) and a candidate transmembrane region (Pro515 Ile534 ) 14,15 (Figure 2). Alignment of the human, mouse and rat heparanase amino acid sequences 123

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The heparanase sequence contains a putative transmembrane domain 15,19 and its complete solubilization from cells requires the presence of a detergent, suggesting that up to 25% of the heparanase activity is membrane associated. 15 In fact, heparanase immunoreactivity was observed on the surface of various human cancer cells, including breast, pancrease and colon carcinomas. 27 The significance of cell surface anchorage and/or binding of heparanase, its activation, cellular uptake and the related effects on cell attachment, migration, metastasis and angiogenesis are being investigated. We have recently cloned a chicken heparanase that, unlike the human enzyme, is readily secreted and preferentially associated with the cell surface. 23 Cells transfected with the chicken enzyme exhibit a higher angiogenic and metastatic potential, as compared with cells overexpressing the human enzyme (Goldshmidt et al., manuscript in preparation). While the involvement of heparanase in cancer metastasis and angiogenesis is well documented, its normal functions were not emphasized. Development of mice with targeted disruption of the heparanase gene is needed to elucidate its normal roles in embryonic development and in the mature individual. Immunolocalization studies preformed in chicken embryos revealed that the heparanase gene and protein are preferentially expressed in cells migrating from the epiblast and forming the hypoblast layer, as early as 12 h post-fertilization. Later on, the protein is highly expressed in the developing nervous and vascular systems. 23 Mammary glands of virgine transgenic mice overexpressing the heparanase enzyme exhibit precocious branching of ducts associated with local dissolution of the underlying BM, suggesting that the enzyme promotes normal morphogenesis and possibly pre-malignant changes in the mammary gland. 28

corresponding to the 50 kDa human mature enzyme (Lys158 -Ile543 ), revealed 80–93% identity. 15 A 61% homology was found between the recently cloned chicken heparanase and the human enzyme. 23 The fact that highly homologous cDNA sequences were derived from different species and types of normal and malignant cells is consistent with the notion that one dominant functional endoglucuronidase is expressed by all mammalian cells. 13–18 Thus, unlike the large number of proteases that can solubilize polypeptides in the ECM, one heparanase appears to be predominantly used by cells to degrade the HS side chains of HSPGs. Secondary structure predictions suggest that heparanase contains a catalytic (α/β) eight TIM-barrel fold (residues 411– 543), characteristic of the clan A glycosyl hydrolase families. 19 Site-directed mutagenesis revealed that, similar to other TIM-barrel glycosyl hydrolases, heparanase has a common catalytic mechanism that involves two conserved acidic residues, a putative proton donor at Glu225 and a nucleophile at Glu343 . Conserved basic residues are found in proximity to the proposed catalytic proton donor and nucleophile 19 (Figure 2).

Regulation and normal function Taking into account the normal functions of heparanase (i.e. morphogenesis, tissue repair, HS turnover, immune surveillance) and because of the potential tissue damage that could result from inadvertent cleavage of HS, tight regulation and balance are essential. Potential regulators of heparanase activity are cytokines, local pH, cellular localization and the apparently membrane bound protease, converting the heparanase from a latent 65 kDa protein into an active 50 kDa form. The nature of this enzyme has not been identified. Factors affecting the cellular localization of heparanase may regulate its availability and biological function. For example, the subcellular localization of cathepsin B in breast and bladder cancer cells changes from within lysosomes to the plasma membrane with increasing metastatic potential. 24 Heparanase activity has been demonstrated in both lysosomal and endosomal compartments in rat ovarian and human colon carcinomas. 25 The enzyme has been localized in perinuclear acidic endosomal granules of fibroblasts and tumour cells (Katz et al., manuscript in preparation) and in the tertiary granules of human neutrophils, co-localized with MMP-9 activity. 12,26

Involvement of heparanase in cancer progression Preferential expression in human tumours Expression of the human heparanase mRNA in normal tissues is restricted primarily to the placenta and lymphoid organs. 8,13–15,29 Real time quantitative RT-PCR revealed increased levels of the heparanase mRNA in human tumours and xenografts of human breast, colon, lung, prostate, ovarian and pancreatic 124

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(a)

(b)

Figure 3. Expression of heparanase in neoplastic human colonic mucosa and stroma. (a) Heparanase immunostaining of tumour tissue (right), and lack of staining in the adjacent normal-looking tissue (left). (b) Heparanase staining of deeply invading tumour cells and of desmoplastic stromal cells surrounding the tumour cells.

tumours that had been propagated in athymic nude mice, compared with the corresponding normal tissues. 29 Expression of the heparanase gene and protein was detected at early stages of human colon carcinoma progression, already at the stage of adenoma, while the adjacent normal-looking colonic tissue showed no expression of the enzyme [Figure 3(a)]. 27 Gradually increasing expression of the enzyme was evident as the cells progressed from severe dysplasia through well differentiated to poorly differentiated colon carcinoma. Deeply invading colon carcinoma cells and desmoplastic stromal fibroblasts in the tumour microenvironment showed high levels of the heparanase mRNA and protein [Figure 3(b)]. 27 Human mammary carcinoma expressed the heparanase mRNA and protein in both the in situ and invasive components of ductal and lobular origins. Intense immunostaining was observed in breast carcinoma cells that have entered the circulation, and in lymph node metastases. 28 Normal breast tissue expressed little or no heparanase. 14,28 Preferential expression of the heparanase mRNA and protein was also clearly

demonstrated in tissue specimens derived from adenocarcinoma of the ovary, metastatic melanoma, oral squamous cell carcinoma, hepatocellular carcinoma and carcinomas of prostate, bladder, intestine and pancreas. 14,27–32 Patients exhibiting high levels of the heparanase mRNA in bladder and pancreatic cancer tissues had a significantly shorter post-operative survival time than patients whose tumours contained relatively low levels of heparanase. 31,32 There is also a significant correlation between enhanced heparanase mRNA expression and tumor vascularity. 31 Angiogenesis and neovascularization HSPGs are prominent components of blood vessels. In large vessels they are concentrated mostly in the intima and inner media, whereas in capillaries they are found mainly in the subendothelial BM where they support proliferating and migrating EC and stabilize the structure of the capillary wall. 33 HSPGs and HSPG-degrading enzymes have long been implicated in a number of angiogenesis-related 125

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Syndecan FGFR bFGF Syndecan FGFR

Heparanase

EC BL

Heparanase

bFGF VEGF

Perlecan

HS

ECM Figure 4. Proposed involvement of heparanase in angiogenesis. Heparanase promotes: (i) EC migration and degradation of the subendothelial basal lamina (BL) and ECM; (ii) release of active HS-bound bFGF and VEGF; and (iii) release of HS degradation fragments that promote FGF-receptor (FGFR) binding, dimerization and signalling (arrows), inducing EC migration and proliferation (reprinted with permission from FASEB J. 15: 1661–1663, 2001).

cellular events, including cell invasion, migration, adhesion, differentiation and proliferation. 3–5,34,35 Heparin and HS sequester, stabilize and protect FGFs and VEGFs from inactivation, and function as low affinity co-receptors that promote dimerization of FGFs and thereby activation of the signalling cell surface tyrosine kinase receptors. 3–7,34,35 An important early step in the angiogenic cascade is degradation of the subendothelial capillary BM by proliferating EC and formation of vascular sprouts. Heparanase, degrading the polysaccharide scaffold of BM, is presumed to facilitate EC invasion and migration toward the angiogenic stimulus (Figure 4), similar to MMPs and other proteolytic enzymes. 36 Heparanase, expressed by platelets, tumour and inflammatory cells, releases active bFGF from ECM and BM as a complex with HS fragment (Figure 4). 6,7 The enzyme also releases bFGF-stimulating HSdegradation fragments from the endothelial cell surface. 7,35 In contrast, HS fragments released from ECM exert little or no potentiation of the growth promoting activity of bFGF. 35 Altogether, it appears that apart from direct involvement in BM invasion by EC, heparanase elicits an indirect angiogenic

response by releasing HS-bound angiogenic growth factors (i.e. bFGF, VEGF) from ECM and BM, and by generating HS fragments which can potentiate bFGF receptor binding, dimerization and signalling (Figure 4). Immunohistochemical staining of human colon, pancrease and breast carcinomas revealed preferential expression of the heparanase protein by EC of sprouting capillaries in the vicinity of the tumour versus little or no staining of mature vessels. 35 Using the mouse matrigel plug angiogenesis assay, we observed an increased angiogenic response to heparanase transfected T lymphoma cells, embedded in matrigel and implanted subcutaneously, versus little or no response to the parental mock transfected cells. 35 Thus, cooperative interactions between heparanases from tumour, inflammation and endothelial sources appear to play a significant role in the angiogenic cascade. Increased tissue vascularity was also observed in a mouse wound-healing model in response to a topical administration of recombinant heparanase. 35 In other studies, MMP-9 was shown to function as a specific component of the angiogenic switch by rendering VEGF more available to its receptors. 37

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inefficiency depends, to a large extent, on the inhibition of growth in a subset of extravasated cells. 39 ECM-degrading enzymes such as MMPs and heparanase may be critical for a proper seeding and proliferation of the disseminated cells through release of sequestered growth and angiogenesis promoting factors, thus creating a more favourable tumour microenvironment.

Cancer metastasis Tumour cell invasion and metastasis involves degradation of ECM constituents through the concerted sequential action of enzymes such as matrix metalloproteinases (MMPs), serine and cysteine proteases, and endoglycosidases. The ability of HSPGs to interact with various ECM macromolecules and with different attachment sites on plasma membranes suggests a key role for this proteoglycan in the self-assembly and insolubility of ECM components, as well as in cell adhesion and locomotion. 1–6 Cleavage of HS may therefore result in disassembly of the subendothelial ECM and play a significant role in extravasation of blood-borne cells. 8–13,21 Expression of heparanase correlates with the metastatic potential of human tumour cells. 9–15 Moreover, elevated levels of heparanase were detected in sera of metastatic tumour bearing animals and cancer patients 9 and in the urine of some patients with aggressive metastatic disease (our unpublished observations). Heparanase-inhibiting molecules (e.g. non-anticoagulant species of heparin, sulphated polysaccharides, polyanionic molecules) reduce the incidence of experimental metastases by more than 90%. 9–11,21,38 Evidence for a direct role of heparanase in tumour metastasis was provided by the conversion of T-lymphoma cells from a non-metastatic to metastatic behaviour following transfection and overexpression of the heparanase gene. 14 A massive liver infiltration of the transfected cells and accelerated mortality of the mice were observed following subcutaneous inoculation of the heparanase overexpressing cells, compared with mice inoculated with mock transfected lymphoma cells. Similarly, transfection of the heparanase gene resulted in an increased lung colonization of intravenously inoculated mouse melanoma cells. 14 Moreover, direct incubation of the latent 65 kDa heparanase enzyme with B16-F1 melanoma cells was associated with cell binding, processing and activation of the enzyme, resulting in a five to 12-fold increase in lung colonization following intravenous inoculation of the cells (Yacoby-Zeevi et al., manuscript in preparation). Intravital videomicroscopy (IVVM) for direct in vivo observation of early steps in metastasis, has recently provided evidence that some of the assumptions about mechanisms of metastasis need to be revised. 39,40 It was proposed that, unlike previous assumptions, extravasation may not be the rate limiting process and that metastatic

Inhibitory molecules Non-anticoagulant species of heparin and various sulphated polysaccharides which inhibited experimental metastasis, also inhibited tumour cell heparanase, while others had little or no effect on both parameters. 9–11,21,38 Regardless of the mode of action, heparin appears to have a beneficial effect in cancer patients. 41 Inhibition of both heparanase activity and experimental metastasis was exerted by heparin species containing >14 sugar units and having sulphate groups at both the N and O positions. 10 While O-desulphation abolished the inhibitory effect of heparin, O-sulphated, Nsubstituted (e.g. N-acetyl or N-hexanoyl) species of low molecular weight heparin retained a high inhibitory activity. 10 Potent inhibition of heparanase activity and tumour metastasis was also obtained with other sulphated polysaccharides (i.e. laminaran sulphate) 11,21,38 and heparin-mimicking polyanionic molecules, although an effect on selectin mediated cell adhesion could not be excluded. 41 The oligosaccharide chain length and degree of sulphation were more important parameters than the sugar composition and type of linkage. Phosphomannopentaose sulphate (PI-88) and maltohexaose sulphate were comparable to heparin in their heparanase inhibiting activity (IC50 1–2 µg ml−1 ). 21 PI- 88 is being evaluated in a multicentre phase II clinical trial. 21 Although heparanaseinhibiting compounds might interfere with normal functions of the enzyme (e.g. immune surveillance, tissue repair, HS turnover), heparanase is an attractive drug target. It is hoped that identification of the sugar residues in HS adjacent to the heparanase cleavage site, as well as crystalization and analysis of the three-dimensional (3D) structure of the enzyme will lead to a rational design of highly specific heparanase inhibitors. Heparanase is the first mammalian HS-degrading enzyme that has been cloned, expressed and characterized. This may lead to identification and cloning of other members of a putative family of 127

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mammalian glycosaminoglycan degrading enzymes (e.g. chondroitinase, dermatanase, keratanase), toward a better understanding of the function and biological significance of both the enzymes and their polysaccharide substrates.

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Acknowledgements 16.

This work was supported by grants from the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities; the Israel Cancer Research Fund; the Association for International Cancer Research, UK; the Mizutani Foundation for Glycosciences; the NIH (R21 CA87085); and the US Army (grant # 0278). The help of Dr Iris Pecker (InSight Ltd.) and InSight Ltd. (Rabin Science Park, Rehovot, Israel) is greatly appreciated.

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References 1. Timple R (1989) Structure and biological activity of basement membrane proteins. Eur J Biochem 180:487–502 2. Kjellen L, Lindahl U (1991) Proteoglycans: structures and interactions. Annu Rev Biochem 60:443–475 3. Bernfield M, Gotte M, Park PW, Reizes O, Fitzgerald ML, Lincecum J, Zako M (1999) Functions of cell surface heparan sulfate proteoglycans. Annu Rev Biochem 68:729–777 4. David G (1993) Integral membrane heparan sulfate proteoglycans. FASEB J 7:1023–1030 5. Iozzo RV (1998) Matrix proteoglycans: from molecular design to cellular function. Annu Rev Biochem 67:609–652 6. Vlodavsky I, Bar-Shavit R, Korner G, Fuks Z (1993) Extracellular matrix-bound growth factors, enzymes and plasma proteins, in Basement Membranes: Cellular and Molecular Aspects (Rohrbach DH, Timpl R, eds) pp. 327–343. Academic Press, Orlando 7. Vlodavsky I, Miao H-Q, Medalion B, Danagher P, Ron D (1996) Involvement of heparan sulfate and related molecules in sequestration and growth promoting activity of fibroblast growth factor. Cancer Metastasis Rev 15:177–186 8. Dempsey LA, Brunn GJ, Platt JL (2000) Heparanase, a potent regulator of cell matrix interactions. TIBS 25:349–355 9. Nakajima M, Irimura T, Nicolson GL (1988) Heparanases and tumor metastasis. J Cell Biochem 36:157–167 10. Vlodavsky I, Moshen M, Lider O, Svahn CM, Ekre HP, Vigoda M, Ishai-Michaeli R, Peretz T (1994) Inhibition of tumor metastasis by heparanase inhibiting species of heparin. Invasion Metastasis 14:290–302 11. Parish CR, Coombe DR, Jakobsen KB, Bennett FA, Underwood PA (1987) Evidence that sulphated polysaccharides inhibit tumour metastasis by blocking tumour-cell-derived heparanases. Int J Cancer 40:511–518 12. Vlodavsky I, Eldor A, Haimovitz-Friedman A, Matzner Y, IshaiMichaeli R, Lider O, Naparstek Y, Cohen IR, Fuks Z (1992) Expression of heparanase by platelets and circulating cells of the immune system: possible involvement in diapedesis and extravasation. Invasion Metastasis 12:112–127 13. Parish CR, Freeman C, Hulett MD (2001) Heparanase: a

21.

22.

23.

24.

25.

26.

27.

28.

29.

128

key enzyme involved in cell invasion. Biochim Biophys Acta 1471:M99–M108 Vlodavsky I et al. (1999) Mammalian heparanase: gene cloning, expression and function in tumor progression and metastasis. Nat Med 5:793–802 Hulett MD, Freeman C, Hamdorf BJ, Baker RT, Harris MJ, Parish CR (1999) Cloning of mammalian heparanase, an important enzyme in tumor invasion and metastasis. Nat Med 5:803–809 Kussie PH, Hulmes JD, Ludwig DL, Patel S, Navarro EC, Seddon AP, Giorgio NA, Bohlen P (1999) Cloning and functional expression of a human heparanase gene. Biochem Biophys Res Commun 261:183–187 Toyoshima MT, Nakajima M (1999) Human heparanase: purification, characterization, cloning, and expression. J Biol Chem 274:24153–24160 Fairbanks MB et al. (1999) Processing of the human heparanase precursor and evidence that the active enzyme is a heterodimer. J Biol Chem 274:29587–29590 Hulett MD, Hornbu JR, Ohms SJ, Zuegg J, Freeman C, Gready JE, Parish CR (2000) Identification of active-site of the pro-metastatic endoglycisidase heparanase. Biochemistry 39:15659–15667 Pikas DS, Li JP, Vlodavsky I, Lindahl U (1998) Substrate specificity of heparanases from human hepatoma and platelets. J Biol Chem 273:18770–18777 Parish CR, Freeman C, Brown KJ, Francis DJ, Cowden WB (1999) Identification of sulfated oligosaccharidebased inhibitors of tumor growth and metastasis using novel in vitro assays for angiogenesis and heparanase activity. Cancer Res 59:3433–3441 Dong J, Kukula AK, Toyoshima M, Nakajima M (2000) Genomic organization and chromosome localization of the newly identified human heparanase gene. Gene 253:171–178 Goldshmidt O, Zcharia E, Aingorn H, Guatta-Rangini Z, Atzmon R, Michal I, Pecker I, Mitrani E, Vlodavsky I (2001) Expression pattern and secretion of human and chicken heparanase are determined by their signal peptide sequence. J Biol Chem 276:29178–29187 Sloane BF, Moin K, Sameni M, Tait LR, Rozhin J, Ziegler G (1994) Membrane association of cathepsin B can be induced by transfection of human breast epithelial cells with c-Ha-ras oncogene. J Cell Sci 107:373–384 Kjellen L, Pertoft H, Oldberg A, Hook M (1985) Oligosaccharides generated by an endoglucuronidase are intermediates in the intracellular degradation of heparan sulfate proteoglycans. J Biol Chem 260:8416–8422 Mollinedo F, Nakajima M, Llorens A, Barbosa E, Callejo S, Gajate C, Fabra A (1997) Major co-localization of the extracellular-matrix degradative enzymes heparanase and gelatinase in tertiary granules of human neutrophils. Biochem J 327:917–923 Friedmann Y, Vlodavsky I, Aingorn H, Aviv A, Peretz T, Pecker I, Pappo O (2000) Expression of heparanase in normal, dysplastic and neoplastic human colon mucosa and stroma. Am J Pathol 157:1167–1175 Zcharia E, Metzger S, Friedmann Y, Pappo O, Aviv A, Elkin E, Pecker I, Peretz T, Vlodavsky I (2001) Molecular properties and involvement of heparanase in cancer progression and mammary gland morphogenesis. Mammary Gland Biol Neoplasm 6:311–322 McKenzie E et al. (2000) Cloning and expression profiling

Involvement of heparanase in cancer progression

30.

31.

32.

33. 34.

35.

36. Stetler-Stevenson WG (1999) Matrix metalloproteinases in angiogenesis: a moving target for therapeutic intervention. J Clin Invest 103:1237–1241 37. Bergers G et al. (2000) Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat Cell Biol 2:737–744 38. Miao HQ, Elkin M, Aingorn E, Ishai-Michaeli R, Stein CA, Vlodavsky I (1999) Inhibition of heparanase activity and tumor metastasis by laminarin sulfate and synthetic phosphorothioate oligodeoxynucleotides. Int J Cancer 83:424–431 39. Chambers AF, Matrisian LM (1997) Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst 89:1260–1270 40. Al-Mehdi AB, Tozawa K, Fisher AB, Shientag L, Lee A, Muschel RJ (2000) Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis. Nat Med 6:100–102 41. Borsig L, Wong R, Feramisco J, Nadeau DR, Varki NM, Varki A (2001) Heparin and cancer revisited: mechanistic connections involving platelets, P-selectin, carcinoma mucins, and tumor metastasis. Proc Natl Acad Sci USA 98: 3352–3357

of hpa2, a novel mammalian heparanase family member. Biochem Biophys Res Commun 276:1170–1177 Ginath S, Menczer J, Friedmann Y, Aingorn H, Aviv A, Tajima K, Dantes A, Glezerman M, Vlodavsky I, Amsredam A (2001) Expression of heparanase, Mdm2, and erbB2 in ovarian cancer. Int J Oncol 18:1133–1144 Gohji K, Hirano H, Okamoto M, Kitazawa S, Toyoshima M, Dong J, Katsuoka Y, Nakajima M (2001) Expression of three extracellular matrix degradation enzymes in bladder cancer. Int J Cancer 95:295–301 Koliopanos A, Friess H, Kleeff J, Shi X, Liao Q, Pecker I, Vlodavsky I, Zimmermann A, Büchler MW (2001) Heparanase expression in primary and metastatic pancreatic cancer. Cancer Res 61:4655–4659 Wight TN (1989) Cell biology of arterial proteoglycans. Arteriosclerosis 9:1–20 Folkman J, Shing Y (1992) Control of angiogenesis by heparin and other sulfated polysaccharides. Adv Exp Med Biol 313:355–364 Elkin M, Ishai-Michaeli R, Friedmann Y, Papo O, Pecker I, Vlodavsky I (2001) Heparanase as mediator of angiogenesis: mode of action. FASEB J 15:1661–1663

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