Báo cáo khoa học: Proteoglycans in health and disease: new concepts for heparanase function in tumor progression and metastasis pptx

MINIREVIEW
Proteoglycans in health and disease: new concepts for
heparanase function in tumor progression and metastasis
Uri Barash
1
, Victoria Cohen-Kaplan
1
, Ilana Dowek
2
, Ralph D. Sanderson
3
, Neta Ilan
1
and
Israel Vlodavsky
1
1 Cancer and Vascular Biology Research Center, Rappaport Faculty of Medicine, Haifa, Israel
2 Department of Otolaryngology, Head and Neck Surgery, Carmel Medical Center, Haifa, Israel
3 Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA
Keywords
C-domain; EGFR; head and neck carcinoma;
heparanase; heparan sulfate; lymph
angiogenesis; MMP; myeloma; signaling;
splice variant
Correspondence
I. Vlodavsky, Cancer and Vascular Research
Center, Rappaport Faculty of Medicine,
Technion, P. O. Box 9649, Haifa 31096,
Israel
Fax: +972 4 8510445
Tel: +972 4 8295410

E-mail:
(Received 7 April 2010, revised 29 June
2010, accepted 1 July 2010)
doi:10.1111/j.1742-4658.2010.07799.x
Heparanase is an endo-b-D-glucuronidase capable of cleaving heparan sul-
fate side chains at a limited number of sites, yielding heparan sulfate frag-
ments of still appreciable size. Importantly, heparanase activity correlates
with the metastatic potential of tumor-derived cells, attributed to enhanced
cell dissemination as a consequence of heparan sulfate cleavage and remod-
eling of the extracellular matrix and basement membrane underlying epithe-
lial and endothelial cells. Similarly, heparanase activity is implicated in
neovascularization, inflammation and autoimmunity, involving the migra-
tion of vascular endothelial cells and activated cells of the immune system.
The cloning of a single human heparanase cDNA 10 years ago enabled
researchers to critically approve the notion that heparan sulfate cleavage by
heparanase is required for structural remodeling of the extracellular matrix,
thereby facilitating cell invasion. Progress in the field has expanded the scope
of heparanase function and its significance in tumor progression and other
pathologies. Notably, although heparanase inhibitors attenuated tumor pro-
gression and metastasis in several experimental systems, other studies
revealed that heparanase also functions in an enzymatic activity-independent
manner. Thus, inactive heparanase was noted to facilitate adhesion and
migration of primary endothelial cells and to promote phosphorylation of
signaling molecules such as Akt and Src, facilitating gene transcription (i.e.
vascular endothelial growth factor) and phosphorylation of selected Src sub-
strates (i.e. endothelial growth factor receptor). The concept of enzymatic
activity-independent function of heparanase gained substantial support by
the recent identification of the heparanase C-terminus domain as the molec-
ular determinant behind its signaling capacity. Identification and character-
ization of a human heparanase splice variant (T5) devoid of enzymatic

activity and endowed with protumorigenic characteristics, elucidation of
cross-talk between heparanase and other extracellular matrix-degrading
enzymes, and identification of single nucleotide polymorphism associated
with heparanase expression and increased risk of graft versus host disease
add other layers of complexity to heparanase function in health and disease.
Abbreviations
ECM, extracellular matrix; EGFR, epidermal growth factor receptor; FGF, fibroblast growth factor; HS, heparan sulfate; HSPGs, heparan
sulfate proteoglycans; HSulf-1, human Sulf1; MMP, matrix metalloproteinase; TIM, triosephosphate isomerase; VEGF, vascular endothelial
growth factor.
3890 FEBS Journal 277 (2010) 3890–3903 ª 2010 The Authors Journal compilation ª 2010 FEBS
Introduction
Proteoglycans are composed of core protein to which
glycosaminoglycan (GAG) side chains are covalently
attached. GAGs are linear polysaccharides consisting
of a repeating disaccharide, generally of an acetylated
amino sugar alternating with uronic acid. Units of
N-acetylglucosamine and glucuronic ⁄ iduronic acid
form heparan sulfate (HS). The polysaccharide chains
are modified at various positions by sulfation, epimer-
ization and N-acetylation, yielding clusters of sulfated
disaccharides separated by low or nonsulfated regions
[1,2]. The sulfated saccharide domains provide numer-
ous docking sites for a multitude of protein ligands,
ensuring that a wide variety of bioactive molecules (i.e.
cytokines, growth factors, enzymes, protease inhibitors,
extracellular matrix proteins) binds to the cell surface
and extracellular matrix (ECM) [3–6] and thereby
functions in the control of normal and pathological
processes, among which are morphogenesis, tissue
repair, inflammation, vascularization and cancer

metastasis [1–3]. Two main types of cell-surface HS
proteoglycan (HSPG) core proteins have been identi-
fied: the transmembrane syndecan with four isoforms,
carrying HS near their extracellular tips and occasion-
ally also chondroitin sulfate chains near the cell sur-
face [3]; and the glycosylphosphatidyl inositol-linked
glypican with six isoforms, carrying several HS side
chains near the plasma membrane and often an addi-
tional chain near the tip of its ectodomain [7]. Two
major types of ECM-bound HSPG are found: agrin,
abundant in most basement membranes, primarily in
the synaptic region [8]; and perlecan, with a wide-
spread tissue distribution and a very complex modular
structure [9]. Accumulating evidences indicate that
HSPGs act to inhibit cellular invasion by promoting
tight cell–cell and cell–ECM interactions, and by main-
taining the structural integrity and self-assembly of the
ECM [10,11]. Notably, one of the characteristics of
malignant transformation is downregulation of GAGs
biosynthesis, especially of the HS chains [10,11]. Low
levels of cell-surface HS also correlate with high meta-
static capacity of many tumors. For example, reduced
syndecan-1 levels on the cell surface of colon, lung,
hepatocellular, breast, and head and neck carcinomas
was associated with increased tumor metastasis [10]. In
other cases, syndecan-1 was nonetheless overexpressed,
and appeared to promote metastasis [12]. This behav-
ior is attributed mostly to HSPGs within the ECM,
exemplified by the protumorigenic function of shed
syndecan-1 in multiple myeloma [10,13] (see below).

In addition to modulation of HSPG levels, expres-
sion of enzymes involved in GAGs biosynthesis and
modification is impaired during cell transformation.
Hereditary multiple exostosis provided the first direct
evidence linking an aberrant HS structure to tumori-
genesis. Hereditary multiple exostosis is an autosomal-
dominant disorder characterized by the presence of
multiple bony outgrowths (exostoses), a consequence
of mutation in EXT family members. These genes
encode an enzyme (GlcA ⁄ GlcNAc transferase)
required for chain elongation and synthesis of HS in
the Golgi apparatus [14,15]. Bone outgrowths as a
result of mutation and inactivation of these enzymes
imply their function as tumor-suppressors. HS can
similarly be modified extracellularly by secreted
enzymes such as heparan sulfate 6-O-endosulfatases
which selectively remove the 6-O-sulfate groups from
HS. Human Sulf-1 (HSulf-1) appears to be misregulat-
ed in cancer; it is present in a variety of normal tissues
but is downregulated in cell lines originating from
ovarian, breast, pancreatic, renal and hepatocellular
carcinomas [16]. Loss of HSulf-1 expression results in
increased sulfation of HSPGs, sustained association of
heparin-binding growth factors with their cognate
receptors and augmented downstream signaling.
Expression of HSulf-1 in cell lines derived from head
and neck carcinoma inhibits cell growth, motility and
invasion in vitro [17]. Similarly, overexpression of
HSulf-1 and HSulf-2 in CAG myeloma cells inhibits
tumor xenograft development and the assembly of

fibroblast growth factor (FGF)-2 signaling complex on
the cell surface [18], supporting its function as negative
regulator of cancer.
Whereas the activity HSulf-1 appeares to attenuate
tumor progression, cleavage of HS by the endo-b-glu-
curonidase heparanase is strongly implicated in cell
dissemination associated with tumor metastasis. Clon-
ing of the heparanase gene 10 years ago [19–22] and
the generation of specific tools (i.e. molecular probes,
antibodies, siRNA) enabled researchers to critically
approve the notion that HS cleavage by heparanase is
required for structural remodeling of the ECM under-
lying tumor and endothelial cells, thereby facilitating
cell invasion [23–25]. Progress in the field and the gen-
eration of genetic tools (i.e. heparanase transgenic and
knockout mice) [26–29] have led in recent years to the
discovery of new concepts which expand the scope
of heparanase function and its significance in tumor
progression and other pathologies.
In this minireview we discuss recent progress in hep-
aranase research, focusing on enzymatic activity-depen-
dent and -independent functions mediated by defined
protein domains and splice variants, and cross-talk
U. Barash et al. New concepts for heparanase function
FEBS Journal 277 (2010) 3890–3903 ª 2010 The Authors Journal compilation ª 2010 FEBS 3891
between heparanase and proteases. Aspects such as
heparanase gene regulation, proteolytic processing, cel-
lular localization and the development of heparanase
inhibitors have been the subject of several recent
review articles [23,25,30,31] and are not discussed in

detail here.
Heparanase in tumor progression and
metastasis
Enzymatic activity capable of cleaving glucuronidic
linkages and releasing polysaccharide chains resistant
to further degradation by the enzyme was first identi-
fied by Ogren & Lindahl [32]. The physiological func-
tion of this activity was initially implicated in the
degradation of macromolecular heparin to physiologi-
cally active fragments [32,33]. The activity of the newly
discovered endo-b-glucuronidase, referred to as hepa-
ranase, was soon after shown to be associated with the
metastatic potential of tumor-derived cells such as B16
melanoma [34] and T-lymphoma [35]. These early
observations gained substantial support when specific
molecular probes became available shortly after clon-
ing of the heparanase gene. Both overexpression and
silencing of the heparanase gene clearly indicate that
heparanase not only enhances cell dissemination, but
also promotes the establishment of a vascular network
that accelerates primary tumor growth and provides a
gateway for invading metastatic cells [23,25]. Although
these studies provided a proof-of-concept for the
prometastatic and proangiogenic capacity of heparan-
ase, the clinical significance of the enzyme in tumor
progression emerged from a systematic evaluation of
heparanase expression in primary human tumors.
Immunohistochemistry, in situ hybridization, RT-PCR
and real time-PCR analyses revealed that heparanase
is upregulated in essentially all human carcinomas

examined [23,25]. Notably, increased heparanase levels
were most often associated with reduced patient sur-
vival post operation, increased tumor metastasis and
higher microvessel density [23–25]. We choose to high-
light the role of heparanase in human cancer by focus-
ing on head and neck carcinoma and multiple
myeloma as examples of solid and hematological
malignancies.
Heparanase in head and neck carcinoma:
signaling in motion
Squamous cell carcinoma of the head and neck contin-
ues to be the sixth most common neoplasm in the
world, with > 500 000 new cases projected annually
[36]. Approximately 200 000 deaths occur yearly as the
result of cancer of the oral cavity and pharynx, and
the outcome has not improved significantly in the past
25 years [37]. Tumor metastases are common among
patients with head and neck cancer with uncontrolled
local or regional disease, and autopsy studies revealed
40–47% overall incidence of distant metastases [38,39].
Applying immunohistochemistry, no staining of hepa-
ranase was detected in normal epithelium adjacent to
the tumor lesions (Fig. 1A), likely due to methylation
of the gene and its repression by p53 [40–43]. By con-
trast, heparanase upregulation was found in the major-
ity of head and neck [44], salivary gland [45], tongue
[46] and oral [47] carcinomas. Notably, respective
patients that exhibit no or weak heparanase staining
are endowed with a favorable prognosis and prolonged
survival post operation [44–46,48]. For example, 70%

of the patients with salivary gland carcinoma that
stained negative for heparanase were still alive
300 months (25 years) following diagnosis, whereas
none of patients stained strongly for heparanase sur-
vived at 300 months [45]. Somewhat surprising, hepa-
ranase upregulation in head and neck and tongue
carcinomas was associated with larger tumors [44,46].
This association was also seen in hepatocellular, breast
and gastric carcinomas [49–51]. Likewise, heparanase
overexpression enhanced [52–55], whereas local deliv-
ery of antiheparanase siRNA inhibited, the progression
of tumor xenografts [56]. These results imply that hep-
aranase function is not limited to tumor metastasis but
is engaged in progression of the primary lesion.
Heparanase and tumor vascularization
The cellular and molecular mechanisms underlying
enhanced tumor growth by heparanase are only start-
ing to be revealed. At the cellular level, both tumor
cells and cells that comprise the tumor microenviron-
ment (i.e. endothelial, fibroblasts, tumor-infiltrating
immune cells) are likely to be affected by heparanase.
The proangiogenic potency of heparanase has been
established clinically [23,25,31] and in several in vitro
and in vivo model systems, including wound healing
[29,57], tumor xenografts [52,55], Matrigel plug assay
[57] and tube-like structure formation [58]. Moreover,
microvessel density was significantly reduced in tumor
xenografts developed by Eb lymphoma cells transfect-
ed with antiheparanase ribozyme [59]. The molecular
mechanism by which heparanase facilitates angiogenic

responses has traditionally been attributed primarily to
the release of HS-bound growth factors such as vascu-
lar endothelial growth factor (VEGF)-A and FGF-2
[60,61], a direct consequence of heparanase enzymatic
activity. In addition, enzymatically inactive heparanase
New concepts for heparanase function U. Barash et al.
3892 FEBS Journal 277 (2010) 3890–3903 ª 2010 The Authors Journal compilation ª 2010 FEBS
was noted to facilitate adhesion and migration of pri-
mary endothelial cells [58] and to promote phosphory-
lation of signaling molecules such as Akt and Src
[53,55,58,62,63], the latter found to be responsible for
VEGF-A induction following exogenous addition of
heparanase or its overexpression [55]. Furthermore,
heparanase was also noted to facilitate the formation
of lymphatic vessels. In head and neck carcinoma, high
levels of heparanase were associated with increased
lymphatic vessel density, increased tumor cell invasion
to lymphatic vessels (Fig. 1B) and increased expression
of VEGF-C [64], a potent mediator of lymphatic vessel
formation [65]. Heparanase overexpression by mela-
noma, epidermoid, breast and prostate carcinoma cells
induced a three- to fivefold elevation of VEGF-C
expression in vitro, and facilitated lymph angiogenesis
of tumor xenografts in vivo, whereas heparanase gene
silencing was associated with decreased VEGF-C levels
[64]. These results suggest that enhanced lymph angio-
genesis by heparanase is not specific for head and neck
carcinoma, but rather is a common trait. Upregulation
of VEGF-C was greatly dependent on the cellular
localization of heparanase. Whereas localization of

heparanase to the cytoplasm (representing secreted
heparanase and predicting poor prognosis of cancer
patients; Fig. 1A, Cyto) was associated with increased
VEGF-C staining, nuclear localization of heparanase
(Fig. 1A, Nuc), shown to correlate with a favorable
prognosis of head and neck cancer patients [44],
was associated with low levels of VEGF-C [64]. Simi-
larly, localization of heparanase in the cell cytoplasm
was associated with activation of the epidermal growth
factor receptor (EGFR) in head and neck carcinoma
[66].
Heparanase and EGFR activation
Decorin, a chondroitin sulfate ⁄ dermatan sulfate pro-
teoglycan directly interacts with EGFR and this evokes
a downregulation of the receptor and inhibition of its
downstream signaling. The antiproliferative effect of
decorin on cancer cells via EGFR is reviewed by
Iozzo & Schaefer [67]. By contrast, EGFR phosphory-
lation is markedly increased in cells overexpressing
Normal
Cyto
Nuc
Hepa
LV
Hepa/LV
A
B
Fig. 1. (A) Immunohistochemical staining of
heparanase in squamous cell carcinoma of
the head and neck (SCCHN) tumor speci-

mens. Formalin-fixed, paraffin-embedded
5 lm sections of head and neck tumors
were subjected to immunostaining of hepa-
ranase, applying anti-heparanase polyclonal
Ig #733. Shown are representative photomi-
crographs of positively stained specimens
exhibiting cytoplasmic (Cyto, middle) and
nuclear (Nuc, lower) heparanase localization.
Normal-looking tissue adjacent to the tumor
lesion stained negative for heparanase
(upper). Nuclear heparanase is associated
with decreased levels of phospho-EGFR,
lower lymph vessel density, and favorable
prognosis of head and neck cancer patients
(see text for details). (B) Heparanase expres-
sion associates with tumor cell invasion into
lymph vessels. Head and neck tumor speci-
men was stained with anti-heparanase poly-
clonal (green, upper) and D2-40 monoclonal
(a marker for human lymphatics; red,
middle) Ig, illustrating heparanase-positive
tumor cells inside a lymphatic vessel lumen
(merge, lower).
U. Barash et al. New concepts for heparanase function
FEBS Journal 277 (2010) 3890–3903 ª 2010 The Authors Journal compilation ª 2010 FEBS 3893
heparanase or following its exogenous addition,
whereas heparanase gene silencing is accompanied by
reduced EGFR and Src phosphorylation levels [66].
Notably, EGFR activation was observed following the
addition or overexpression of mutated, enzymatically

inactive heparanase protein. Although inactive, dou-
ble-mutated (Glu225, Glu343) [68] heparanase retains
its high affinity towards HS and hence may facilitate
signaling by ligation and activation of membrane
HSPGs such as syndecan [69,70]. This however
appears not to be the case because heparanase deleted
for its heparin-binding domain (D10) [71] efficiently
stimulated EGFR phosphorylation [66]. Notably,
enhanced EGFR phosphorylation by heparanase was
restricted to selected tyrosine residues (i.e. 845, 1173)
thought to be direct targets of Src rather than a result
of receptor autophosphorylation [72]. Indeed,
enhanced EGFR phosphorylation of tyrosine residues
845 and 1173 in response to heparanase was abrogated
in cells treated with Src inhibitors or antiSrc siRNA
[66]. The functional significance of EGFR modulation
by heparanase emerged by monitoring cell prolifera-
tion. Thus, heparanase gene silencing was accompanied
by a decrease in cell proliferation, whereas heparanase
overexpression resulted in enhanced cell proliferation
and the formation of larger colonies in soft agar, in a
Src- and EGFR-dependent manner [66]. The clinical
relevance of the heparanase–Src–EGFR pathway has
been elucidated for head and neck carcinoma. Nota-
bly, heparanase expression in head and neck carcino-
mas correlated with phospho-EGFR immunostaining,
and even more significant was the correlation between
heparanase cellular localization (i.e. cytoplasmic versus
nuclear) and phospho-EGFR levels [66]. These studies
provide a more realistic view of heparanase function in

the course of tumor progression. Thus, while heparan-
ase enzymatic activity has traditionally been implicated
in tumor metastasis, the current view points to a multi-
faceted protein engaged in multiple aspects of tumor
progression, combining enzymatic activity-dependent
and -independent activities of heparanase and affecting
two systems critical for tumor progression, namely
tumor vascularization and EGFR activation.
Signaling by the heparanase C-domain
The concept of enzymatic activity-independent func-
tion of heparanase gained substantial support by the
recent identification of the heparanase C-domain as
the molecular determinant behind its signaling capac-
ity. The existence of a C-terminus domain (C-domain)
emerged from a prediction of the 3D structure of a
single-chain heparanase enzyme [73]. In this protein
variant, the linker segment was replaced by three gly-
cine–serine repeats (GS3), resulting in a constitutively
active enzyme [74]. The structure obtained clearly illus-
trates a triosphosphate isomerase (TIM)-barrel fold, in
agreement with previous predictions [68,75]. Notably,
the structure also delineates a C-terminus fold posi-
tioned next to the TIM-barrel fold [73]. The predicted
heparanase structure led to the hypothesis that the
seemingly distinct protein domains observed in the 3D
model, namely the TIM-barrel and C-domain regions,
mediate enzymatic and nonenzymatic functions of hep-
aranase, respectively. Interestingly, cells transfected
with the TIM-barrel construct (amino acids 36–417)
failed to display heparanase enzymatic activity, sug-

gesting that the C-domain is required for the establish-
ment of an active heparanase enzyme, possibly by
stabilizing the TIM-barrel fold [73]. Deletion and site-
directed mutagenesis further indicated that the
C-domain plays a decisive role in heparanase enzy-
matic activity and secretion [73,76,77]. Notably, Akt
phosphorylation was stimulated by cells overexpressing
the C-domain (amino acids 413–543), whereas the
TIM-barrel protein variant yielded no Akt activation
compared with control, mock-transfected cells [73].
These findings clearly indicate that the nonenzymatic
signaling function of heparanase leading to activation
of Akt is mediated by the C-domain. Notably, the
C-domain construct lacks the 8 kDa segment (Gln36–
Ser55) which, according to the predicted model,
contributes one beta strand to the C-domain structure
(reviewed in [78]). Indeed, Akt phosphorylation was
markedly enhanced and prolonged in cells transfected
with a mini gene comprising this segment linked to the
C-domain sequence (8-C) [73,78]. This finding further
supports the predicted 3D model, indicating that the
C-domain is indeed a valid functional domain respon-
sible for Akt phosphorylation. The cellular conse-
quences of C-domain overexpression were best
revealed by monitoring tumor xenograft development.
Remarkably, tumor xenografts produced by
C-domain-transfected glioma cells grew faster and
appeared indistinguishable from those produced by
cells transfected with the full-length heparanase in term
of tumor size and angiogenesis, yielding tumors sixfold

bigger than control. By contrast, progression of tumors
produced by TIM-barrel-transfected cells appeared
comparable with control mock-transfected cells [73,78].
These results show, that in some tumor systems (i.e.
glioma), heparanase facilitates primary tumor progres-
sion regardless of its enzymatic activity, whereas in
others (i.e. myeloma) heparanase enzymatic activity
dominates (see below). Enzymatic activity-independent
function of heparanase is further supported by the
New concepts for heparanase function U. Barash et al.
3894 FEBS Journal 277 (2010) 3890–3903 ª 2010 The Authors Journal compilation ª 2010 FEBS
recent identification of T5, a functional human splice
variant of heparanase.
T5, a functional human heparanase splice variant
Almost all protein-coding genes contain introns that
are removed in the nucleus by RNA splicing and are
often alternatively spliced. Alternative splicing
increases the coding capacity of the genome, generat-
ing multiple proteins from a single gene. The resulting
protein isoforms frequently exhibit different biological
properties that may play an essential role in tumori-
genesis [79,80]. A splice variant of human heparanase
which lacks exon 5 has been described [81,82]. This
splice variant fails to get secreted and lacks enzymatic
activity and its biological significance remains unclear.
Additional human heparanase splice variants have
been predicted in silico [83]; the expression of one,
termed T5 (Fig. 2A), was found to be enriched in lung
carcinoma and chronic myeloid leukemia compared
with control tissue and cells. In this splice variant,

144 bp of intron 5 are joined with exon 4, resulting in
a 169-amino-acids protein that lacks the enzymatic
activity typical of heparanase [83]. Unlike previously
identified splice variants of heparanase, T5 is secreted
and facilitates Src phosphorylation [83]. Furthermore,
Src phosphorylation was markedly reduced in cells
treated with antiT5 siRNA [83]. Overexpression of T5
by pharynx (FaDu), myeloma (CAG) and embryonic
kidney (293) cells resulted in enhanced proliferation
and larger colony formation in soft agar, which was
attenuated by Src inhibitor (Fig. 2B) [83]. Likewise, T5
gene silencing was associated with reduced cell prolifer-
ation, indicating that endogenous levels of T5 and hep-
aranase affect tumor cell proliferation. Moreover,
development of tumor xenografts produced by hepa-
ranase- and T5-infected myeloma cells was markedly
enhanced compared with xenografts generated by con-
trol cells (Fig. 2C) [83]. Tumors developed by
T5-expressing cells exhibited a higher density of blood
vessels decorated with smooth muscle actin-positive
cells (pericytes) [83], an indication of vessel matura-
tion. The clinical relevance of T5 emerged from analy-
sis of renal cell carcinoma biopsies, in which T5 and
heparanase expression appeared to be induced in 75%
of cases [83]. Thus, although inhibitors directed against
the enzymatic activity of heparanase are being cur-
rently evaluated in clinical trials [84–87], T5 and the
heparanase C-domain are not expected to be affected
by these inhibitors. It appears, therefore, that a well-
defined enzymatic activity thought to be relatively easy

to target, turned, at least in certain tumor systems,
into a complex objective as more knowledge accumu-
lates and the biology of the protein is being elucidated.
SP 8

kDa
linker
158–
166
SKK
T5
EE
W.T
SP
8

kDa linker
50 kDa
225 343
158–543
110–157
36–1091–35
A
Vo
Hepa
T5
B
Vo Hepa T5
CAG
FaDu

293
DMSO
PP2
C
Fig. 2. Heparanase splice variant, T5, endowed with protumorigenic characteristics. (A) Schematic structure of wild-type (WT) and heparan-
ase splice variant, T5. SP-signal peptide; glutamic acids residues 225 and 343 critical for heparanase enzymatic activity, are detonated (see
text for details). (B) Colony formation in soft agar. Control (Vo) heparanase (Hepa)-, and T5-infected myeloma (CAG, upper), pharynx (FaDu,
second panels) and embryonic kidney (293, third panels) cells (5 · 10
3
cellsÆdish
)1
) were mixed with soft agar and cultured for 3–5 weeks.
CAG cells were similarly grown in the absence (dimethylsulfoxide; fourth panels) or presence of Src inhibitor (PP2, 0.4 n
M; lower panels).
Shown are representative photomicrographs of colonies at high (·100) magnification. (C) Tumor xenograft development. Control (Vo), hepa-
ranase-, and T5-infected CAG myeloma cells were injected subcutaneously (1 · 10
6
0.1 mL
)1
). At the end of the experiment on day 37,
tumors were harvested and photographed.
U. Barash et al. New concepts for heparanase function
FEBS Journal 277 (2010) 3890–3903 ª 2010 The Authors Journal compilation ª 2010 FEBS 3895
Multiple myeloma: moving antiheparanase
therapy closer to reality
Multiple myeloma is the second most prevalent hema-
tologic malignancy. This B-lymphoid malignancy is
characterized by tumor cell infiltration of the bone
marrow, resulting in severe bone pain and osteolytic
bone disease. Although progress in the treatment of

myeloma patients has been made over the last decade,
the overall survival of patients is still poor.
Heparanase enzymatic activity was elevated in the
bone marrow plasma of 86% of myeloma patients
examined [88], and gene array analysis showed ele-
vated heparanase expression in 92% of myeloma
patients [89]. Heparanase upregulation in myeloma
patients was associated with elevated microvessel
density and syndecan-1 expression [88]. Although
heparanase is proangiogenic in myeloma, which is
a common feature shared with solid tumors, hepa-
ranase regulation of syndecan-1 shedding has
emerged as highly relevant to multiple myeloma
progression.
Syndecan-1 is particularly abundant in myeloma,
and is the dominant and often the only HSPG pres-
ent on the surface of myeloma cells [90]. Cell-surface
syndecan-1 promotes adhesion of myeloma cells and
inhibits cell invasion in vitro [13]. By contrast, high
levels of shed syndecan-1 are found in the serum of
some myeloma patients and are associated with poor
prognosis [91]. The multiple roles of syndecans in
cancer progression and strategies for their targeting
is presented in the accompanying minireview by
Theocharis et al. [92]. Shed syndecan-1 becomes
trapped within the bone marrow ECM where it likely
acts to enhance the growth, angiogenesis and metas-
tasis of myeloma cells within the bone [13,93,94].
This is supported by the finding that enhanced
expression of soluble syndecan-1 by myeloma cells

promotes tumor growth and metastasis in a mouse
model [13,94]. Notably, heparanase upregulates both
the expression and shedding of syndecan-1 from the
surface of myeloma cells [89,95]. In agreement with
this notion, heparanase gene silencing was associated
with decreased levels of shed syndecan-1 [89]. Impor-
tantly, both syndecan-1 upregulation and shedding
require heparanase enzymatic activity, because over-
expression of mutated inactive heparanase failed to
stimulate syndecan-1 expression and shedding [95].
Syndecan-1 shedding was similarly augmented by the
addition of recombinant active heparanase to CAG
myeloma cells, and even more dramatic shedding was
observed following the addition of bacterial heparin-
ase III (heparitinase) [95]. These findings indicate that
cleavage of HS by heparanase or heparinase III may
render syndecan-1 more susceptible to proteases
mediating the shedding of syndecan-1. However, it
appears that heparanase may play an even more
direct role in regulating shedding of syndecan-1, by
facilitating the expression of proteases engaged in
syndecan shedding.
Heparanase–matrix metalloproteinase cooperation in
myeloma progression
It was recently demonstrated that enhanced expres-
sion of heparanase leads to increased levels of matrix
metalloproteinase (MMP)-9 (a syndecan-1 sheddase),
whereas heparanase gene silencing resulted in reduced
MMP-9 activity [96]. Upregulation of MMP-9 expres-
sion has significant biological relevance because inhi-

bition of MMP-9 reduces syndecan-1 shedding [96].
For the importance of syndecan shedding in diseases
see the accompnaying minireview by Manon-Jensen
et al. [97]. Moreover, not only MMP-9, but also uro-
kinase-type plasminogen activator and its receptor,
molecular determinants responsible for MMP-9 acti-
vation, are upregulated by heparanase. These findings
provided the first evidence for cooperation between
heparanase and MMPs in regulating HSPGs on the
cell surface and likely in the ECM, and are supported
by the recent generation and characterization of hepa-
ranase knockout mice. HS chains isolated from these
mice were longer, critically supporting the notion that
heparanase is the only functional endoglycosidase
capable of degrading HS [26]. Despite the complete
lack of heparanase gene expression and enzymatic
activity, heparanase knockout mice develop normally,
are fertile and exhibit no apparent anatomical or
functional abnormalities [26]. Interestingly, heparanase
deficiency was accompanied by a marked elevation of
MMP family members such as MMP-2, MMP-9 and
MMP-14, in an organ-dependent manner. Thus,
MMP-14 levels were increased eightfold in the liver
of heparanase knockout mice compared with control
littermates, whereas MMP-2 levels were increased 2.5-
fold in the mammary gland [26], suggesting that
MMPs provide tissue-specific compensation for
heparanase deficiency. This is likely the reason for
over-branching of the mammary gland in heparanase-
knockout mice [26], a phenotype also noted in

heparanase transgenic mice [27]. Collectively, these
results suggest that heparanase is intimately engaged
in the regulation of gene transcription and acts as a
master regulator of protease expression, mediating
gene induction or repression, depending on the
biological setting.
New concepts for heparanase function U. Barash et al.
3896 FEBS Journal 277 (2010) 3890–3903 ª 2010 The Authors Journal compilation ª 2010 FEBS
The heparanase–syndecan axis is a target for therapy
Results from studies using several in vivo model sys-
tems support the notion that enzymatic activities
responsible for syndecan-1 modification are valid tar-
gets for myeloma therapy. For example, enhanced
expression of either HSulf-1 or HSulf-2 attenuated
myeloma tumor growth [18]. Even a more dramatic
inhibition of tumor growth was noted following
administration of bacterial heparinase III (heparitin-
ase) to SCID mice inoculated with either CAG mye-
loma cells or cells isolated from the bone marrow of
myeloma patients [98]. Although heparinase III and
human heparanase both degrade HS chains, their
cleavage products are distinct. Whereas heparinase III
is a b-eliminase that extensively degrades HS, heparan-
ase is an endo-b-d-glucuronidase whose substrate-rec-
ognition sites were recently characterized [99]. Unlike
the bacterial enzyme, heparanase cleaves HS more
selectively and generates fragments of 4–7 kDa, yield-
ing strictly distinct outcomes in the context of tumor
progression. Although administration of heparinase III
is associated with reduced tumor growth, heparanase

activity is elevated in many hematological and solid
tumors, correlating with poor prognosis and shorter
post-operative survival rate (see above). Accordingly,
inhibition of heparanase enzymatic activity is expected
to suppress tumor progression. To examine this in
myeloma, a chemically modified heparin, which is
100% N-acetylated and 25% glycol-split was tested.
This flexible molecule is a potent inhibitor of heparan-
ase enzymatic activity, lacks anticoagulant activity typ-
ical of heparin, and does not displace ECM-bound
FGF-2 or potentiate its mitogenic activity
[30,31,100,101]. The modified heparin profoundly
inhibits the progression of tumor xenografts produced
by myeloma cells [30,98]. These studies support the
notion that heparanase enzymatic activity not only
facilitates tumor metastasis, but also promotes the pro-
gression of primary tumors.
Conclusions and perspective
Although much has been learned in the last decade,
the repertoire of heparanase functions in health and
disease is only starting to emerge. Clearly, from activ-
ity implicated mainly in cell invasion associated with
tumor metastasis, heparanase has turned into a multi-
faceted protein that appears to participate in essen-
tially all major aspects of tumor progression. In this
regard, evidence now supports a concept by which
growth of the primary tumor is fueled by circulating
metastatic tumor cells [102,103]. According to this
notion, tumor cells are present in the circulation in
large numbers even at the early stages of cancer and

long before metastatic growth at distant sites can be
detected [103]. These cells can reinfiltrate and promote
growth and angiogenesis of the primary tumor [102].
The possible involvement of heparanase in tumor self-
seeding is supported by the timing of its induction dur-
ing tumorigenesis and its prometastatic function. Using
the RIP-Tag2 tumor model, it was demonstrated that
heparanase mRNA and protein are elevated upon the
transition from normal to angiogenic islets, followed
by a further increase when solid tumors were detected
[104]. Furthermore, heparanase expression is elevated
already at the early stages of human neoplasia. In the
colon, heparanase gene and protein are expressed
already at the stage of adenoma [105], and during esoph-
ageal carcinogenesis heparanase expression is induced in
Barrett’s epithelium (Fig. 3), an early event that predis-
poses patients to the formation of dysplasia which may
progress to adenocarcinoma [106]. Tumor self-seeding
also facilitates the recruitment of stromal components.
Although the proangiogenic capacity of heparanase has
been established, its likely impact on other components
of the tumor microenvironment (i.e. fibroblasts, macro-
phages) awaits thorough investigation.
Heparanase expression at the early stages of tumor
initiation and progression, and by the majority of
tumor cells (evident by a high extent of immunostain-
ing), can be utilized to turn the immune system against
the very same cells. Accumulating evidence suggests
that peptides derived from human heparanase can eli-
cit a potent antitumor immune response, leading to

lysis of heparanase-positive human gastric
(KATO III), colon (SW480) and breast (MCF-7) carci-
noma cells, as well as hepatoma (HepG2) and sarcoma
(U-2 OS) cells [107–109]. By contrast, no killing effect
was noted towards autologous lymphocytes [107–109].
Notably, the development of tumor xenografts pro-
duced by B16 melanoma cells was markedly restrained
in mice immunized with peptides derived from mouse
heparanase (i.e. amino acids 398–405; 519–526) com-
pared with a control peptide in both immunoproection
and immunotherapy approaches [109]. T-regulatory
cells are frequently present in colorectal cancer
patients. Interestingly, T-regulatory cells against hepa-
ranase could not be found [110]. Antiheparanase
immunotherapy is thus expected to be prolonged and
more efficient due to the absence of T-suppressor cells.
A related treatment approach is being tested in
advanced metastasized breast cancer patients [111].
Although this immunotherapeutic concept, together
with available heparanase inhibitors, is hoped to
advance cancer treatment, the identification of single
U. Barash et al. New concepts for heparanase function
FEBS Journal 277 (2010) 3890–3903 ª 2010 The Authors Journal compilation ª 2010 FEBS 3897
nucleotide polymorphism associated with heparanase
expression and increased risk for graft versus host dis-
ease following allogeneic stem cell transplantation
[112–114] offers a genetic concept which can potentially
be translated into patients’ diagnosis. Studies in these
directions, identification of heparanase receptor(s)
mediating its signaling function, and elucidation of

heparanase route and function in the cell nucleus, will
advance the field of heparanase research and reveal its
significance in health and disease. Resolving the hepa-
ranase crystal structure will accelerate the development
of effective inhibitory molecules and neutralizing anti-
bodies paving the way for advanced clinical trials in
patients with cancer and other diseases (i.e. colitis, pso-
riasis, diabetic nephropathy) involving heparanase.
Acknowledgements
We thank Prof. Benito Casu (‘Ronzoni’ Institute,
Milan, Italy) for his continuous support and active
Heparanase Ki-67
Normal
Barrett
Low
dysplasia
High
dysplasia
Carcinoma
Fig. 3. Immunohistochemical staining of
esophageal specimens. Formalin-fixed,
paraffin-embedded 5 lm sections of normal
(upper panel), Barrett’s (second panel),
low-grade (third panel), high-grade (fourth
panel) and adenocarcinoma (lower panel)
esophageal biopsies were subjected to
immunostaining of heparanase, applying
anti-heparanase polyclonal Ig #733 (left
panels) or anti-(Ki-67), a marker of cell
proliferation (right panels).

New concepts for heparanase function U. Barash et al.
3898 FEBS Journal 277 (2010) 3890–3903 ª 2010 The Authors Journal compilation ª 2010 FEBS
collaboration. This work was supported by grants
from the Israel Science Foundation (grant 549 ⁄ 06);
National Institutes of Health (NIH) grants CA138535
(RDS) and CA106456 (IV); the Israel Cancer Research
Fund (ICRF); and the Juvenile Diabetes Research
Foundation (JDRF grant 1-2006-695). I. Vlodavsky is
a Research Professor of the ICRF. We gratefully
acknowledge the contribution, motivation and assis-
tance of the research teams in the Hadassah-Hebrew
University Medical Center (Jerusalem, Israel) and the
Cancer and Vascular Biology Research Center of the
Rappaport Faculty of Medicine (Technion, Haifa). We
apologize for not citing several relevant articles, due to
space limitation.
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