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

Abstract: Heparanase is an endo-β-glucuronidase that cleaves heparan sulfate side chains presumably at sites of low sulfation, activity that is strongly implicated with cell invasion associated with cancer metastasis, a consequence of structural modification that loosens the extracellular matrix barrier. In addition, heparanase exerts pro-adhesive properties, mediated by clustering of membrane heparan sulfate proteoglycans (i.e., syndecans) and activation of signaling molecules such as Akt, Src, EGFR, and Rac in a heparan sulfate-dependent and -independent manner. Activation of signaling cascades by enzymatically inactive heparanase and by a peptide corresponding to its substrate binding domain not only increases cell adhesion but also facilitates cancer cell growth. This notion is supported by preclinical and clinical settings, encouraging the development of anti-heparanase therapeutics. Here, we summarize recent progress in heparanase research emphasizing the molecular mechanisms that govern its pro-tumorigenic and pro-adhesive properties. Pro-adhesive properties of the heparanase homolog, heparanase 2 (Hpa2), are also discussed. Enzymatic activity-independent function of proteases (i.e., matrix metalloproteinases) is discussed in the context of cell adhesion and tumor progression. Collectively, these examples suggest that enzyme function exceeds beyond the enzymatic aspect, thus significantly expanding the scope of the functional proteome. Cross-talk with matrix metalloproteinases and the role of heparanase in pathological settings other than cancer are also described. [ABSTRACT FROM AUTHOR]

Abstract: The endoglycosidase heparanase is the predominant enzyme that degrades heparan sulfate side chains of heparan sulfate proteoglycans, activity that is strongly implicated in tumor metastasis. Apart of its well characterized enzymatic activity, heparanase was noted to exert also enzymatic-independent functions. Among these is the induction of Akt/PKB phosphorylation noted in endothelial- and tumor-derived cells. Protein domains of heparanase required for signaling were not identified to date, nor were identified heparanase binding proteins/receptors capable of transmitting heparanase signals. Here, we examined the possible function of mannose 6-phosphate receptor (MPR) and low-density lipoprotein-receptor related protein (LRP), recently implicated in cellular uptake of heparanase, as heparanase receptors mediating Akt phosphorylation. We found that heparanase addition to MPR- and LRP-deficient fibroblasts elicited Akt activation indistinguishable from control fibroblasts. In contrast, disruption of lipid rafts abrogated Akt/PKB phosphorylation following heparanase addition. These results suggest that lipid raft-resident receptor mediates heparanase signaling. [Copyright &y& Elsevier]

Although cancer cells use heparanase for tumor metastasis, favourable effects of heparanase have been reported in the management of Alzheimer's disease and diabetes. Indeed, we previously established a protective function for heparanase in the acutely diabetic heart, where it conferred cardiomyocyte resistance to oxidative stress and apoptosis by provoking changes in gene expression. In this study, we tested if overexpression of heparanase can protect the heart against chemically induced or ischemia/reperfusion (I/R) injury. Transcriptomic analysis of Hep-tg hearts reveal that 240 genes related to the stress response, immune response, cell death, and development were altered in a pro-survival direction encompassing genes promoting the unfolded protein response (UPR) and autophagy, as well as those protecting against oxidative stress. The observed UPR activation was adaptive and not apoptotic, was mediated by activation of ATF6α, and when combined with mTOR inhibition, induced autophagy. Subjecting wild type (WT) mice to increasing concentrations of the ER stress inducer thapsigargin evoked a transition from adaptive to apoptotic UPR, an effect that was attenuated in Hep-tg mouse hearts. Consistent with these observations, when exposed to I/R, the infarct size and markers of apoptosis were significantly lower in the Hep-tg heart compared to WT. Finally, UPR and autophagy inhibitors reduced the protective effects of heparanase overexpression during I/R. Our data suggest that the mechanisms that underlie the role of heparanase in promoting cell survival could be uniquely beneficial to the heart by providing protection against cellular stresses, and could be useful for exploitation as a therapeutic target for the treatment of heart disease. Unlabelled Image • Cardiac overexpression of heparanase upregulated pro-survival and catabolism genes. • These gene changes contributed to activation of adaptive UPR and autophagy. • Activation of ATF6α is a key initiator of adaptive UPR. • Heparanase promotes autophagy by UPR activation and mTOR inhibition. • These adaptations protected the heart against chemical and I/R induced injury. [ABSTRACT FROM AUTHOR]

Vaccinia virus (VACV), like many other viruses, binds to cell surface heparan sulfate (HS) prior to infecting cells. Since HS is ubiquitously expressed extracellularly, it seemed likely that VACV-HS interaction may impede virus spread, with host heparanase, the only known mammalian endoglycosidase that can degrade HS, potentially overcoming this problem. In support of this hypothesis, we found that, compared to wild type, mice deficient in heparanase showed a 1–3 days delay in the spread of VACV to distant organs, such as ovaries, following intranasal inoculation, or to ovaries and spleen following intramuscular inoculation. These delays in spread occurred despite heparanase deficiency having no effect on VACV replication at inoculation sites. Subsequent in vitro studies revealed that heparanase treatment released VACV from HS expressing, but not HS deficient, infected cell monolayers. Collectively these data suggest that VACV relies on host heparanase to degrade HS in order to spread to distant sites. • Poxviruses, like several other viruses, use cell surface heparan sulfate (HS) as a co-receptor during infection. • Heparanase is the only known mammalian endoglycosidase that can degrade HS. • Spread of HS binding viruses like vaccinia virus (VACV), is aided by host derived heparanase. • Heparanase treatment releases VACV from HS expressing, but not HS deficient, cell monolayers infected with VACV. • Targetting heparanase could be a novel approach to controlling HS-dependent viral infection. [ABSTRACT FROM AUTHOR]

Abstract: Heparanase is an endoglycosidase that cleaves heparan sulfate in the extracellular matrix (ECM) and hence participates in ECM degradation and remodeling. Heparanase is involved in fundamental biological processes such as cancer metastasis, angiogenesis, and inflammation. Alternative splicing in the coding region of human heparanase was not reported. Here, we report the cloning of a splice variant of human heparanase that lacks exon 5 and is missing 174bp compared to the wild-type cDNA. Splice 5 is expressed as a 55kDa protein compared to the 65 and 50kDa latent and active wild-type enzyme. Splice 5 was not detected in the incubation medium of tumor cells as opposed to the wild-type latent heparanase. Splice 5 escaped proteolytic cleavage, was devoid of HS degradation activity and exhibited diffused rather than granular cellular localization. [Copyright &y& Elsevier]

Abstract: Objective. : To analyze the expression of Heparanase, an enzyme involved in cancer metastasis and angiogenesis, in ovarian and breast carcinoma cells in effusions. Methods. : Heparanase protein expression was analyzed in malignant effusions from ovarian (=200) and breast (=41) carcinoma patients using immunocytochemistry. The levels of secreted heparanase were analyzed in 45 effusion supernatants using a newly established ELISA test. Heparanase expression levels were analyzed for clinical significance. Results. : Heparanase was expressed at the cell membrane in 106/200 (53%) ovarian and 22/41 (54%) breast carcinomas. Cytoplasmic expression was found in 180/200 (90%) ovarian and 26/41 (63%) breast carcinomas. Reactive mesothelial cells showed frequent cytoplasmic, but not membrane expression. ELISA showed secreted heparanase in all 45 analyzed effusions. Higher levels were detected in peritoneal compared to pleural effusions (p =0.031). In univariate survival analysis of ovarian carcinoma patients with post-chemotherapy effusions, membrane expression in >5% of tumor cells correlated with shorter overall survival (OS, p =0.013). FIGO stage (p =0.03 for all patients, p =0.045 for those with post-chemotherapy specimens) and response to first-line chemotherapy (p <0.0001 for all patients, p =0.049 for those with post-chemotherapy specimens) were the clinical parameters related to OS. In Cox analysis of this subset of patients, heparanase expression (p =0.02) and response to chemotherapy (p =0.049) were independent predictors of poor OS. Heparanase expression did not correlate with survival in breast carcinoma. Conclusions. : Our data show that heparanase is frequently expressed in metastatic gynecologic adenocarcinomas, and that it is secreted into the effusion fluid in body cavities. The correlation between heparanase expression and poor survival in ovarian carcinoma suggests a role for this molecule in ovarian cancer metastasis and supports its role as a marker of aggressive clinical behavior at disease recurrence. [Copyright &y& Elsevier]

Abstract: Heparanase is a mammalian endo-β-d-glucuronidase that cleaves heparan sulfate side chains at a limited number of sites. Heparanase enzymatic activity is thought to participate in degradation and remodeling of the extracellular matrix and to facilitate cell invasion associated with tumor metastasis, angiogenesis, and inflammation. Traditionally, heparanase activity was well correlated with the metastatic potential of a large number of tumor-derived cell types. More recently, heparanase upregulation was detected in an increasing number of primary human tumors, correlating, in some cases, with poor postoperative survival and increased tumor vascularity. The present study was undertaken to develop a highly sensitive ELISA suitable for the determination and quantification of human heparanase in tissue extracts and body fluids. The assay preferentially detects the 8+50kDa active heparanase heterodimer vs. the latent 65kDa proenzyme and correlates with immunoblot analysis of heparanase containing samples. It detects heparanase at concentrations as low as 200pg/ml and is suitable for quantification of heparanase in tissue extracts and urine. [Copyright &y& Elsevier]

Abstract: Heparanase is a heparan sulfate-degrading endoglycosidase expressed by mature monocytes and myeloid cells, but not by immature hematopoietic progenitors. Heparanase gene expression is upregulated during differentiation of immature myeloid cells. PML-RARα and PLZF-RARα fusion gene products associated with acute promyelocytic leukemia abrogate myeloid differentiation and heparanase expression. AML-Eto, a translocation product associated with AML FAB M2, also downregulates heparanase gene expression. The common mechanism that underlines the activity of these three fusion gene products involves the recruitment of histone deacetylase complexes to specific locations within the DNA. We found that retinoic acid that dissociates PML-RARα from the DNA, and which is used to treat acute promyelocytic leukemia patients, restores heparanase expression to normal levels in an acute promyelocytic leukemia cell line. The retinoic acid effects were also observed in primary acute promyelocytic leukemia cells and in a retinoic acid-treated acute promyelocytic leukemia patient. Histone deacetylase inhibitor reverses the downregulation of heparanase expression induced by the AML-Eto fusion gene product in M2 type AML. In summary, we have characterized a link between leukomogenic factors and the downregulation of heparanase in myeloid leukemic cells. [Copyright &y& Elsevier]

Heparanase is an endo-β-d-glucuronidase involved in cleavage of heparan sulfate residues and hence participates in extracellular matrix degradation and remodeling. The heparanase cDNA encodes for a polypeptide of 543 amino acids that appears as a ∼65 kDa band in SDS–PAGE analysis. The protein undergoes a proteolytic cleavage that is likely to occur at two potential cleavage sites, Glu109–Ser110 and Gln157–Lys158, yielding an 8 kDa polypeptide at the N-terminus, a 50 kDa polypeptide at the C-terminus, and a 6 kDa linker polypeptide that resides in-between. The active form of heparanase has long been thought to be a 50 kDa polypeptide isolated from cells and tissues. However, attempts to obtain heparanase activity after expression of the 50 kDa polypeptide failed, suggesting that the N-terminal region is important for heparanase enzymatic activity. It has been hypothesized that heterodimer formation between the 8 and 50 kDa heparanase subunits is important for heparanase enzymatic activity. By individually or co-expressing the 8 and 50 kDa heparanase subunits in mammalian cells, we demonstrate specific association between the heparanase subunits by means of co-immunoprecipitation and pull-down experiments. Moreover, a region in the 50 kDa heparanase subunit that mediates interaction with the 8 kDa subunit was identified. Altogether, our results clearly indicate that heterodimer formation is necessary and sufficient for heparanase enzymatic activity in mammalian cells. [Copyright &y& Elsevier]

Heparanase is an endo-β-d-glucuronidase involved in degradation of heparan sulfate (HS) and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues. The enzymatic activity of heparanase is characterized by specific intrachain cleavage of glycosidic bonds with a hydrolase mechanism. This enzyme facilitates cell invasion and hence plays a role in tumor metastasis, angiogenesis, inflammation, and autoimmunity. Although the expression pattern and molecular properties of heparanase have been characterized, its subcellular localization has not been unequivocally determined. We have previously suggested that heparanase subcellular localization is a major determinant in regulating the enzyme''s biological functions. In the present study we examined heparanase localization in three different cell types, utilizing immunofluorescent staining and electron microscopy. Our results indicate that heparanase is localized primarily within lysosomes and the Golgi apparatus. A construct composed of heparanase cDNA fused to green fluorescent protein, utilized in order to visualize the enzyme within living cells, confirmed its localization in acidic vesicles. We suggest that following synthesis, heparanase is transported into the Golgi apparatus and subsequently accumulates in a stable form within the lysosomes, where it functions in HS turnover. The lysosomal compartment may also serve as a site for heparanase confinement within the cells, limiting its secretion and uncontrolled extracellular activities associated with tumor metastasis and angiogenesis. [Copyright &y& Elsevier]