Sanderson3, Neta Ilan1and Israel Vlodavsky1 1 Cancer and Vascular Biology Research Center, Rappaport Faculty of Medicine, Haifa, Israel 2 Department of Otolaryngology, Head and Neck Surg
Trang 1Proteoglycans in health and disease: new concepts for
heparanase function in tumor progression and metastasis Uri Barash1, Victoria Cohen-Kaplan1, Ilana Dowek2, Ralph D Sanderson3, Neta Ilan1and
Israel Vlodavsky1
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: vlodavsk@cc.huji.ac.il
(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 sulsul-fate 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 endotheepithe-lial 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.
Trang 2Proteoglycans 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 -indepenactivity-depen-dent functions mediated by defined protein domains and splice variants, and cross-talk
Trang 3between 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
Trang 4was 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
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 invaexpres-sion into
lymph vessels Head and neck tumor
speci-men was stained with anti-heparanase
poly-clonal (green, upper) and D2-40 monopoly-clonal
(a marker for human lymphatics; red,
middle) Ig, illustrating heparanase-positive
tumor cells inside a lymphatic vessel lumen
(merge, lower).
Trang 5heparanase 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
Trang 6recent 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 sgenerat-ingle gene The resultgenerat-ing
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–
166SKK
T5
W.T SP 8 kDa linker 50 kDa
225 343 158–543 110–157
36–109 1–35
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 heparanheparan-ase 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.
Trang 7Multiple 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 (heparitinheparin-ase) [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
Trang 8The 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-glucuronidheparan-ase 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 Usdur-ing 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] Antihepahepa-ranase 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
Trang 9nucleotide 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).
Trang 10collaboration 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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