Heparanase—A single protein with multiple enzymatic and nonenzymatic functions

Abstract Heparanase (Hpa1) is expressed by tumor cells and cells of the tumor microenvironment and functions extracellularly to remodel the extracellular matrix (ECM) and regulate the bioavailability of ECM‐bound factors, augmenting, among other effects, gene transcription, autophagy, exosome formation, and heparan sulfate (HS) turnover. Much of the impact of heparanase on tumor progression is related to its function in mediating tumor‐host crosstalk, priming the tumor microenvironment to better support tumor growth, metastasis, and chemoresistance. The enzyme appears to fulfill some normal functions associated, for example, with vesicular traffic, lysosomal‐based secretion, autophagy, HS turnover, and gene transcription. It activates cells of the innate immune system, promotes the formation of exosomes and autophagosomes, and stimulates signal transduction pathways via enzymatic and nonenzymatic activities. These effects dynamically impact multiple regulatory pathways that together drive tumor growth, dissemination, and drug resistance as well as inflammatory responses. The emerging premise is that heparanase expressed by tumor cells, immune cells, endothelial cells, and other cells of the tumor microenvironment is a key regulator of the aggressive phenotype of cancer, an important contributor to the poor outcome of cancer patients and a valid target for therapy. So far, however, antiheparanase‐based therapy has not been implemented in the clinic. Unlike heparanase, heparanase‐2 (Hpa2), a close homolog of heparanase (Hpa1), does not undergo proteolytic processing and hence lacks intrinsic HS‐degrading activity, the hallmark of heparanase. Hpa2 retains the capacity to bind heparin/HS and exhibits an even higher affinity towards HS than heparanase, thus competing for HS binding and inhibiting heparanase enzymatic activity. It appears that Hpa2 functions as a natural inhibitor of Hpa1 regulates the expression of selected genes that maintain tissue hemostasis and normal function, and plays a protective role against cancer and inflammation, together emphasizing the significance of maintaining a proper balance between Hpa1 and Hpa2.

and plays a protective role against cancer and inflammation, together emphasizing the significance of maintaining a proper balance between Hpa1 and Hpa2.  Table 1.
Heparan sulfate proteoglycans (HSPGs) are a fundamental class of extracellular matrix (ECM) constituents, comprised of pericellular or extracellular core proteins conjugated to one or more chains of the glycosaminoglycan polysaccharide HS. 119 HSPGs mediate myriad biological processes, including signal transduction, developmental patterning, 120 cell adhesion, 121 barrier formation, 122 endocytosis 123 and viral entry. 95,124 These processes largely depend upon the HS polysaccharides adorning the core protein, whose heterogeneous structure allows interaction with multiple partners. Given their heterogeneity and versatility, HSPGs serve as important functional components of the cell surface, glycocalyx, and ECM. Hence, cleavage of HS by heparanase affects a diverse and expanding repertoire of physiological and pathological processes. The enzyme appears to fulfill some normal functions, associated, for example, with vesicular traffic, lysosomal-based secretion, autophagy, tissue remodeling, HS turnover, and gene transcription. [10][11][12][13] It activates cells of the innate immune system, promotes the formation of exosomes and autophagosomes, and stimulates signal transduction pathways via enzymatic and nonenzymatic activities. [10][11][12][13]82 These effects dynamically impact multiple regulatory pathways that together drive tumor growth, dissemination, and drug resistance as well as inflammatory responses. 2,[11][12][13] A key venue by which heparanase accomplishes its multiple effects on cells and tissues is by regulating the bioavailability of HS-bound growth factors, chemokines, and cytokines. In this way, heparanase mediates tumor-host crosstalk and promotes basic cellular processes that together orchestrate tissue remodeling. 82 Among the proteins sequestered by the ECM are typical proangiogenic mediators such as platelet-derived growth factor, hepatocyte growth factor (HGF), basic fibroblast growth factor, heparin-binding epithelial growth factor, and vascular endothelial growth factor A (VEGF-A). 29,30,125,126 Release of these proteins by heparanase contributes to the strong proangiogenic response observed in preclinical models and clinical settings. 13,31,[127][128][129][130] HISTORY Activity capable of cleaving macromolecular heparin at a limited number of sites was first reported in mastocytoma cells. 4 Soon thereafter, Höök et al. 131 reported an endoglycosidase activity that degrades HS glycosaminoglycans into oligosaccharides. Attempts to purify the enzyme yielded some misleading results, culminating, nearly 10 years later, in purification of the platelet enzyme 7 and cloning of a single human heparanase complementary DNA sequence, independently by four groups. [32][33][34][35] Given the structural role of HSPGs in the assembly of the ECM and basement membrane, it was hypothesized that HS-degrading activity will loosen the ECM, thus promoting cell dissemination. Indeed, early on, heparanase activity was found to correlate with the metastatic potential of tumor cells, 8,9,132 a correlation that still directs and guides heparanase research. Studies performed before cloning of the HPSE gene contributed immensely to our understanding of key features in the biology of the enzyme, its mode of action, and involvement in cancer metastasis and inflammation. 133,134 Soon after cloning of the HPSE gene and the development of antiheparanase antibodies and probes, many studies examined its expression in human tumors compared with the adjacent normal tissue. Immunohistochemistry, in situ hybridization, realtime-polymerase chain reaction and enzymatic activity analyses revealed that heparanase is upregulated in essentially all human tumors examined. 2,[11][12][13][14]128,130,135 In contrast, the normal-looking tissue adjacent to the malignant lesion expresses little or no detectable levels of heparanase, indicating that fibroblasts and epithelial cells do not normally express the enzyme. The molecular mechanisms underlying heparanase induction in tumor cells are not entirely clear but involve epigenetic alterations (i.e., DNA methylation), hormones, oncogenes, and transcriptional/posttranscriptional regulation by elements (3ʹ-UTR, enhancer, insulator) that activate or suppress the HPSE promoter. 12,45,136 Selected observations are referred to in Table 1.
Clinically, patients that were diagnosed as heparanase-positive exhibited a significantly higher rate of local and distant metastases as well as reduced postoperative survival, compared with patients that were diagnosed as heparanase-negative. [15][16][17]137 These and more recent studies [18][19][20][21][22][23] provide strong clinical support for the and other genes, 66,89,90,128,142,[144][145][146] thus significantly expanding its functional repertoire and mode of action in promoting tissue inflammation and aggressive tumor behavior. 96 Several excellent up-to-date reviews describe basic and translational aspects of heparanase. 11,12,96 This review is aimed at further increasing awareness of this multifaceted protein, highlighting the significance of the Hpa1-heparanase-2 (Hpa2) axis and addressing obstacles in implementing antiheparanase therapies.

NONENZYMATIC ACTIVITIES
Years before the resolution of the heparanase crystal structure, Fux et al. 72 predicted the structure of enzymatically active, single-chain, heparanase enzyme, in which the linker segment was replaced by three glycine-serine repeats (GS3), resulting in a constitutively active enzyme. 37 The structure clearly illustrated a C-terminus (═C-domain) fold positioned next to the TIM-barrel structure. 72  active site. 1 This inactive form of heparanase was found to exert epidermal growth factor receptor (EGFR) phosphorylation and Akt phosphorylation. 80 It was also reported that enzymatically quiescent heparanase augmented T-cell interactions with VCAM-1 and ECM components. 148 Notably, heparanase enhances platelet adhesive capacity and thrombogenicity 149 and also supports the clustering of circulating tumor cells 150 thereby contributing to the metastatic cascade, largely independent of its enzymatic activity. Likewise, the upregulation of HGF, MCP-1, and TF expression in response to heparanase was independent of its enzyme activity. 145,151,152 Moreover, heparanase enhances the phosphorylation of selected signaling molecules including Akt, Src, and EGFR, which in turn facilitates STAT3 phosphorylation, in a manner that requires secretion but not enzymatic activity of heparanase, evident by being mediated by the enzyme C-terminus domain and by the inactive double mutant protein 72,80,81 (Table 1). A later observation of integrindependent phosphoinositide 3-kinase (PI3K)/Akt activation in response to heparanase further highlighted the nonenzymatic activity of heparanase in promoting signal transduction. 153 Notably, the ability of heparanase to activate PI3K/Akt in a nonenzymatic manner, essentially bypassing PTEN signaling, is evidence of its ability to counter tumorsuppressive mechanisms. 11,153 The results suggest that in certain cells, heparanase activates SRC family kinase in an enzymatically independent manner which proceeds to stimulate EGFR. EGFR can then activate downstream pathways such as PI3K-Akt and mitogen-activated protein kinase-extracellular signal-regulated kinase, possibly resulting in phosphorylation of signal transducer and activator of transcription 3 (STAT3) which then drives tumorigenic responses. 10,128

SIGNAL TRANSDUCTION
Heparanase interacts with syndecans by virtue of their HS content and the typical high affinity that exists between the enzyme and its substrate. This high-affinity interaction directs the clustering of syndecans followed by rapid and efficient uptake of heparanase 56,154 ( Figure 1). Mechanistically, syndecan clustering by heparanase or the KKDC peptide, corresponding to the heparin-binding domain of F I G U R E 1 Schematic presentation of heparanase and Hpa2 biosynthesis and trafficking. Pre-proheparanase (red circles) and Hpa2 (blue triangles) are first targeted to the ER lumen via their own signal peptides (1). The proteins are then shuttled to the Golgi apparatus and are subsequently secreted via vesicles that bud from the Golgi (2). Once secreted, heparanase rapidly interacts with syndecans, resulting in their clustering and signaling (3), followed by rapid endocytosis of the heparanase-syndecan complex (5) that accumulates in late endosomes (6). Hpa2 interacts with cell membrane HSPG (i.e., syndecans) with higher affinity but unlike heparanase, is not subjected to uptake but rather remains on the cell membrane for a relatively long period of time (4 and left inset). Accumulation of Hpa2 in the extracellular compartment is enhanced by heparin or anti-Hpa2 monoclonal antibody. Heparanase uptake is inhibited by heparin/heparin mimetics, antiheparanase monoclonal antibodies, or Hpa2, resulting in extracellular accumulation of the latent enzyme (5). Conversion of endosomes to lysosomes (6) results in heparanase processing and activation (primarily by cathepsin L) awaiting secretion (7). Typically, heparanase appears in perinuclear lysosomes (right inset), promoting autophagy (8) and tumor growth, metastasis, angiogenesis, and chemoresistance due to its enzymatic and signaling (9) functions. Hpa2, on the other hand, attenuates tumor growth and vascularity. Novel heparanase inhibitors are expected to target extracellular latent (signaling) and active heparanase as well as the intracellular, lysosomal, enzyme. ER, endoplasmic reticulum; Hpa2, heparanase-2; HSPG, heparan sulfate proteoglycan. PROTEOGLYCAN RESEARCH | 5 of 12 heparanase, 64 enhance cell adhesion and spreading, associated with PKC, Src, and Rac1 activation, 155 molecular determinants shown to be induced by syndecans. [156][157][158] Cell adhesion represents a nonenzymatic signaling function of heparanase in its simplest term. 159,160 Heparanase was also noted to elicit signaling in a manner that does not involve HS. Signaling is considered to be HSindependent if it occurs in HS-deficient cells (i.e., CHO-745) or in the presence of heparin, as demonstrated for enhanced Akt phosphorylation by heparanase. 161 Heparin, a potent competitive inhibitor of heparanase enzymatic activity, when added together with heparanase, augmented, rather than attenuated, Akt phosphorylation, 161 critically implying that heparanase enzymatic activity is not required for Akt activation. Importantly, in xenograft models, heparanase overexpression resulted in tumors bigger in size 45,162 coupled with increased Akt phosphorylation. 45,72 Adversely, heparanase gene silencing was associated with reduced Akt phosphorylation. 140,163 Related studies revealed that heparanase stimulates the phosphorylation of STAT3, STAT5, Src, EGFR, Erk, and the insulin receptor and also activates G-protein receptor signaling, 65,80,81,164 all function to promote tumorigenesis ( Figure 1 and Table 1).

DNA DAMAGE
Associations between heparanase and various pathologies, including inflammation and cancer metastasis, have historically been attributed to the cleavage of HS chains at the cell surface and basement membrane; however, as discussed above, heparanase may possess unique roles arising independently of its enzymatic active site. For example, while the enzymatic activity of heparanase appears to enable viral release through splitting of HS residues at the cell surface, heparanase was reported to also regulate gene expression and trigger proviral signaling through some distinct nonenzymatic activity. 97 It appears that heparanase acts beyond its established endoglycosidase activity as a potent regulator of the signal transduction phase of cellular defense. In this respect, it was reported that cells lacking heparanase display enhanced sensitivity to DNA damage-induced death 96 and are intrinsically resistant to herpes simplex virus-1 infection. 96 Moreover, the interferon system is constitutively enhanced in the absence of heparanase and deletion of heparanase protects against cellular infiltration and associated inflammation. 96,97,165 Repeat immunopurifications of heparanase showed robust binding of proteins heavily implicated in DNA damage sensing and repair, suggesting that heparanase plays a significant role as a regulator of DNA damage response signals. 96

NUCLEAR HEPARANASE
Nuclear HS inhibits histone acetyltransferases (HATs), and thereby gene transcription. 73 Heparanase contains two potential nuclear localization sequences, and enzymatically active heparanase has been found in the chromatin compartment of the nucleus where it colocalizes with RNA polymerase II and positively controls the transcription of genes important for T cells' immune function. 79 Likewise, by entering the nucleus and degrading nuclear syndecan-1, heparanase mediates HAT activation and transcription of genes associated with an aggressive tumor phenotype. 73 Conversely, nuclear heparanase binds nonspecifically to DNA and competes for binding with nuclear factor-κB (NF-κB), thus preventing transcription of NF-κB target genes and acting as a tumor suppressor. 166 At the molecular level, nuclear heparanase appears, among other effects, to regulate histone 3 lysine 4 methylation by influencing the recruitment of demethylases to transcriptionally active genes. 79 Together it appears that nuclear heparanase promotes chromatin remodeling that opens its conformation allowing access to promotors of genes that affect cancer progression. 167,168 In-depth research is still needed to better elucidate the mode of heparanase nuclear translocation and transcriptional activity. 73,79

HEPARANASE-INHIBITING COMPOUNDS
To date, only four compounds have progressed to clinical trials. 10,41,[91][92][93]129 These four "best-in-class" inhibitors are all polyanionic oligo-/polysaccharides that mimic physicochemical properties of the natural heparanase inhibitor heparin, a glycosaminoglycan related to HS. The heparin-like properties of these inhibitors, along with their structural heterogeneity, likely produce unwanted pleiotropic effects (e.g., anticoagulation, growth factor binding, poor pharmacokinetics) that complicate their clinical use. The active site of heparanase has proven challenging for small-molecule pharmacological intervention, given its extensive interaction surface evolved to bind large HS polysaccharides. 169 Such challenging sites are often well targeted by mechanism-based covalent inhibitors, which may still react with an enzyme despite weak or transient initial binding.
Indeed, HS-configured pseudodisaccharide, designed to bind selectively to the heparanase active site and react with its essential catalytic nucleophile, was recently synthesized. 169 This nanomolar, mechanism-based, irreversible heparanase inhibitor markedly reduced cancer aggression in in cellulo and in vivo cancer models. 169 Recently, chemoenzymatic synthesis of sulfur-linked sugar polymers was reported to yield potent competitive heparanase inhibitors. 170 Importantly, heparanase knockout (KO) mice exhibit no obvious immunological and other deficits, 53 implying that inhibition of heparanase will cause minimal side effects in cancer patients.
Dual and apparently antagonistic effects of antiheparanase therapy should be considered. 11 For example, it was reported that heparanase plays a critical role in natural killer (NK) cell invasion into tumors. 100 It was shown that cytokine and immune checkpoint blockade immunotherapy for metastases were compromised when NK cells lacked heparanase. 100 Likewise, it was found that in contrast to freshly isolated T lymphocytes, HPSE mRNA is downregulated in in vitro-expanded T cells. This may explain the reduced ability of cultured CAR-T cells to penetrate stroma-rich solid tumors compared with lymphoid tissues. Indeed, engineering the CAR-T cells to express HPSE resulted in their improved capacity to degrade the ECM, which promoted tumor T cell infiltration and antitumor activity. 171 It was suggested that the use of this strategy might enhance the activity of CAR-T cells in individuals with stroma-rich solid tumors. These results should be considered when systemically treating cancer patients with heparanase inhibitors since the potential adverse effect on NK and CAR-T cells on cell infiltration might limit the antitumor activity of the inhibitors. 171 It appears that targeting the tumor microenvironment by heparanase inhibitors enhances the antitumor activity of approved therapies, further providing a strong rationale for applying antiheparanase therapy in combination with conventional anticancer drugs. 141 Remarkably, heparanase inhibitors were effective even when the xenografted tumor cells were devoid of heparanase, emphasizing the significance of heparanase contributed by cells residing in the tumor microenvironment. 88

HPA2-A NEW PLAYER IN THE HEPARANASE FIELD
HPSE2, the gene encoding Hpa2, was cloned soon after the cloning of heparanase, based on sequence homology. 101 Hpa2 gained attention when it was found that the HPSE2 gene is mutated in a human disease called urofacial syndrome (UFS). 102,103 UFS is a rare autosomal recessive congenital disease, featuring a combination of urological defects and an inverted facial expression attributed to peripheral neuropathy. 104 Notably, Hpa2 lacks intrinsic HS-degrading activity, the hallmark of heparanase, 105 yet it retains the capacity to interact with HS. 105 Hpa2 exhibits an even higher affinity towards heparin and HS than heparanase, 105 114 Collectively, these results support the notion that Hpa2 functions as a tumor suppressor ( Figure 2 and Table 1). Heparanase and Hpa2 not only exhibit opposite functions in terms of tumor growth but also in terms of the underlying mechanism. For example, while heparanase induces VEGF-A and VEGF-C 142 expression and promotes angiogenesis, Hpa2 attenuates the expression of VEGF-A and VEGF-C and decreases tumor vascularity 106 ; whereas heparanase attenuates cell differentiation and promotes epithelial-to-mesenchymal transition (EMT), 172,173 Hpa2 increases cell differentiation. 106,111 This mirrored functionality suggests that Hpa2 exerts these properties in part by modulating heparanase, as demonstrated by a significant decrease in heparanase activity in sarcoma cells overexpressing Hpa2. 108

CONCLUSIONS AND PERSPECTIVES
Among other aspects, heparanase research reinforced the significance of the ECM in the control of cell proliferation and differentiation. 174,175 It led to important and often unexpected observations in diverse normal and pathological processes including, wound healing, 176 angiogenesis, 31 autophagy, 99 signal transduction, 72,97,128,153 protein trafficking, 177 lysosomal secretion, 129  progression. 10,72,189,190 Based on the housekeeping nature of its gene promoter, we suggest that heparanase is expressed at low levels by all cells, modulating autophagy and possibly other functions of the lysosome.
According to this notion, heparanase function in the lysosome may not be less important than its function extracellularly. This may turn relevant in platelets, neutrophils, lymphocytes, and macrophages that show relatively high levels of heparanase expression and activity. 11,12,33,90,191 Beyond serving as a cellular recycling center, recent evidence suggests that the lysosome is involved in homeostasis, generating building blocks for cell growth, mitogenic signaling, angiogenesis, metastasis, and activation of transcriptional programs, 192,193 repertoire that closely resembles those of heparanase.
The PI3-kinase/Akt/mammalian target of rapamycin (mTOR) is highly implicated in the regulation of cell metabolism, protein homeostasis, and cell growth due, in part, to the localization of mTOR at the lysosome membrane which is required for its activation. 194,195 Indeed, Akt is the most common kinase activated by heparanase, 72,80,140,153,161,163 and its instrumental role in the regulation of mTOR would likely convey to the lysosome. 194,195 Research is

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.

ETHICS STATEMENT
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