Human Heparanase Research Articles

Regulation of mouse Heparanase gene expression in T lymphocytes and tumor cells

Heparanase (HPSE) is an endoglycosidase that cleaves heparan sulfate (HS) and plays an important role in tumor metastasis, angiogenesis and inflammation. The regulation of HPSE expression and function is tightly controlled and the increasing use of the mouse as an animal model to define the role of HPSE in many physiological and pathological settings, makes understanding the regulatory mechanisms of HPSE in this species of fundamental importance. However, the expression distribution of the mouse Hpse gene and the mechanisms that regulate its transcription are poorly defined. In this study, the mouse Hpse gene was determined to encode for two mRNA transcripts of 1.9 and 3.2 kb in length with identical open reading frames that showed similar tissue expression distribution to the human HPSE. The mouse Hpse promoter was cloned and a 478-bp minimal promoter was identified that contained regulatory elements responsible for both basal promoter activity in mouse tumor cells as well as inducible activity in T cells. Mutagenesis and transactivation studies identified a functional site in the minimal promoter region for the transcription factor Early growth response gene 1 (Egr1). Interestingly, Egr1 acted differentially in mouse tumor cells, functioning in an activating or repressive manner in breast carcinoma or melanoma cells, respectively. Furthermore, the proximal region of the promoter, identified as important in the regulation of Hpse transcription, was shown to become accessible in T cells upon cell activation. Significantly, the maximal accessibility of the promoter occurred at 16 h post-stimulation, which correlated with the induction kinetics of Hpse mRNA expression. In summary, this study demonstrates that mouse Hpse is expressed and regulated in a similar manner to human HPSE and also provides some novel insights into mechanisms of Hpse gene regulation that are likely to be relevant to control of the human gene.

Cloning, expression, and characterization of an alternatively spliced variant of human heparanase

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 174 bp compared to the wild-type cDNA. Splice 5 is expressed as a 55 kDa protein compared to the 65 and 50 kDa 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.

Structure modeling, ligand binding, and binding affinity calculation (LR‐MM‐PBSA) of human heparanase for inhibition and drug design

Human heparanase is an endo-beta-D-glycosidase that cleaves heparan sulphate (HS) chains in the extracellular matrix and basement membrane. It is known that the cleavage of HS by heparanase results in cell invasion and metastasis of cancer. Therefore, heparanase is considered an important target for cancer drug development. The three-dimensional structure of heparanase would be useful in the rational design of inhibitors targeted to the enzyme; however, the three-dimensional structure has not yet been determined. In our effort to design inhibitors, we developed a three-dimensional structure of heparanase using a homology-modeling approach. The homology-built structure is consistent to previous bioinformatics and site-mutation experimental results. The heparanase features a (alpha/beta)(8) TIM-barrel fold with two glutamate residues (Glu225 and Glu343) located in the active-site cleft. This feature supports the putative mechanism of proton donor and nucleophilic sites. Docking simulations yielded 41 complex structures, which indicate that the bound inhibitor could block ligand binding into the catalytic site. A free energy of binding model was established for 25 heparanase inhibitors with a training set of 25 heparanase inhibitors using the linear response MM-PBSA approach (LR-MM-PBSA). The correlation between calculated and experimental activity was 0.79 and the reliability of the model was validated with leave-one-out cross-validation method. Its predictive capability was further validated using a test set of 16 inhibitors similar to the training set of inhibitors. The correlation between the predicted and observed activities is significantly improved by the protein "induced-fit" that accounts for the flexibility of the receptor. These interaction and pharmacophore elements provide a unique insight to the rational design of new ligands targeted to the enzyme.

Heparanase is expressed in osteoblastic cells and stimulates bone formation and bone mass

Heparan sulfate proteoglycans (HSPGs) are ubiquitous macromolecules. In bone, they are associated with cell surfaces and the extracellular matrix (ECM). The heparan sulfate (HS) chains of HSPGs bind a multitude of bioactive molecules, thereby controlling normal and pathologic processes. The HS-degrading endoglycosidase, heparanase, has been implicated in processes such as inflammation, vascularization associated with wound healing and malignancies, and cancer metastasis. Here we show progressive mRNA expression of the hpa gene (encoding heparanase) in murine bone marrow stromal cells undergoing osteoblastic (bone forming) differentiation and in primary calvarial osteoblasts. Bone marrow stromal cells derived from transgenic mice expressing recombinant human heparanase (rh-heparanase) and MC3T3 E1 osteoblastic cells exposed to soluble rh-heparanase spontaneously undergo osteogenic differentiation. In addition, the transgenic bone marrow stromal cells degrade HS chains. In wild-type (WT) and hpa-transgenic (hpa-tg) mice, heparanase is weakly expressed throughout the bone marrow with a substantial increase in osteoblasts and osteocytes, especially in the hpa-tg mice. Heparanase expression was absent in osteoclasts. Micro-computed tomographic and histomorphometric skeletal analyses in male and female hpa-tg versus WT mice show markedly increased trabecular bone mass, cortical thickness, and bone formation rate, but no difference in osteoclast number. Collectively, our data suggest that proteoglycans tonically suppress osteoblast function and that this inhibition is alleviated by HS degradation with heparanase.

An ELISA method for the detection and quantification of human heparanase

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 + 50 kDa active heparanase heterodimer vs. the latent 65 kDa proenzyme and correlates with immunoblot analysis of heparanase containing samples. It detects heparanase at concentrations as low as 200 pg/ml and is suitable for quantification of heparanase in tissue extracts and urine.

Spotlight

Reisinger et al., pp. 1603–1608 Epstein-Barr Virus (EBV) preferentially infects B cells and is associated with benign and malignant diseases. In this study, Reisinger et al. established a means of visualizing the genomic organization of the virus in infected cells. Using a technique based on dynamic molecular combing (DMC), DNA from Burkitt's lymphoma cell lines, Daudi and Namalwa—which have episomal and integrated EBV DNA, respectively—were probed with EBV-specific DNA probes and visualized using fluorescence in situ hybridization (FISH). When the authors examined the Namalwa DNA, all 54 FISH signals displayed a split terminal repeat (TR) sequence pattern, which strongly suggests that the virus has been integrated in these cells. On the other hand, when Daudi cells were subjected to the same DNA probes, all 59 hybridization patterns visualized displayed a single—unsplit—TR signal, which is the hallmark of episomal expression. This technique seems to unambiguously distinguish between episomal and integrated forms of EBV DNA. Importantly it could be used to further investigate how EBV DNA is habored in various diseases and whether integrated forms are associated with oncogenic consequences. Cohen et al., pp. 1609–1617 Heparanase is the main enzyme that cleaves heparin sulfate, an abundant polysaccharide in the extracellular matrix (ECM). Its activity has been demonstrated in a host of malignancies, and is known to be important in metastasis and tumor angiogenesis. Previous studies have shown that heparanase expression in breast cancer patients correlates with the metastatic potential of the tumor. In this report, Cohen et al. investigated the role of the enzyme in primary breast cancer progression. To do this, they stably (and mock) transfected a breast cancer cell line, MCF-7, with human heparanase (H-hpa) cDNA and injected these cells into the mammary pads of nude mice. Tumor growth after 30 days—as monitored by MRI—showed that heparanase expression accelerated MCF-7 tumor growth in vivo. Not only that, but the resultant tumors' volume and mass were 7 times greater in the H-hpa fashioned tumors than in the mock transfected ones. Futhermore, ex vivo the H-hpa-expressing MCF-7 tumors had a higher mitotic activity and proliferation rate and lower apoptotic potential. This study clearly shows that heparanase both affects primary breast cancer growth and offers insight into why cells expressing this enzyme might have a survival advantage. Bak et al., pp. 1618–1622 DNA adducts—complexes that form when a chemical binds to DNA—have been shown to be necessary but insufficient for tumorigenesis. Human carcinogens have been isolated in such adducts. Using a Danish population cohort comprising over 50,000 people with no previous cancer diagnosis, the authors investigated whether levels of bulky DNA adducts in white blood cells (WBC) are a reliable risk indicator of lung cancer. Using 32P-postlabeling, Bak et al. analyzed WBC bulky DNA in 245 study participants who developed lung cancer and 255 healthy controls randomly selected and stratified by age, sex and duration of smoking. Current smokers with high—above the median—levels of bulky adducts had a higher risk (1.61 95% CI 1.04-2.49) of lung cancer than those with adduct levels below the median. In comparison with previous studies, Bak and colleagues' comprehensive study is population based and investigates adducts prior to cancer diagnosis. Their findings indicate a weak association between bulky DNA adducts in WBC risk and lung cancer risk. Davidson et al., pp. 1623–1627 Circadian rhythms are known to be important for biological processes as fundamental as cell division. Recent data suggest that disruption of circadian rhythmicity can affect tumor growth in rodents. In this report, Davidson et al. investigated whether circadian rhythms were distinct in hepatocellular carcinoma cells (HCC) compared to healthy liver tissue and whether these processes could be disrupted by experimental restricted feeding (RF) or jet-lag. Rats transgenic for a luciferase reporter gene driven by core circadian clock gene, Period 1, were chemically induced to develop HCC and exposed to different restricted feeding patterns—including night-time and ad libitum feeds (n=18)—or experimental jet-lag (n=20). The authors then explanted the hepatomas and normal liver tissue and measured Period 1 activity with bioluminescence. Restricted feeding—both day or night—altered Period 1 expression in both malignant and healthy cells. Interestingly, the HCC cells were much less sensitive to this effect: peak Period 1 expression in HCC in day-fed vs. night-fed groups differed by 6.3 hours, whereas in healthy liver it differed by over 11 hours. This differential circadian rhythmicity found between cancerous and healthy tissue could be exploited to use restricted feeding to target cancer cells with chemotherapy when the tumors cells are in a mitotic phase and healthy cells not.

Heparanase Upregulates Synthesis and Expression of Syndecan-1 (CD138) - A Novel Growth Regulatory Loop for Myeloma Tumors.

Syndecan-1 is a transmembrane heparan sulfate-bearing proteoglycan expressed on the surface of most myeloma tumor cells as well as some other tumors (e.g., breast cancer). The extracellular domain of syndecan-1 is shed from the surface of tumor cells by proteolytic enzymes and accumulates in the extracellular matrix and in the blood. High levels of soluble syndecan-1 in the blood are an indicator of poor prognosis for myeloma patients. Human heparanase-1 (heparanase) is an enzyme that releases biologically active fragments of heparan sulfate chains. In addition, growth factors within the tumor microenvironment that are bound to heparan sulfate are released by heparanase activity. In previous in vivo studies we demonstrated that enhanced expression of heparanase or soluble syndecan-1 by myeloma cells dramatically increases tumor growth and upregulates their spontaneous metastasis. We have now discovered that an increase in heparanase expression on tumor cells leads to enhanced expression, shedding and accumulation of syndecan-1 within the tumor microenvironment. One myeloma cell line and two breast cancer cell lines transfected with the cDNA for heparanase exhibit a dramatic increase in shed syndecan-1 as compared to equal number of control cells that were transfected with empty vector (2.7-fold, 6.3-fold and 17-fold increase over controls, respectively). This accumulation of syndecan-1 in the culture media was not accompanied by an increase in cell surface syndecan-1 levels as assessed by flow cytometry. Gene array analysis demonstrates that following transfection of the myeloma cell line with heparanase, the expression of the syndecan-1 gene is upregulated 1.4-fold. Together these findings suggest that expression of heparanase elevates syndecan-1 transcription and rate of shedding from the cell surface. To examine this further, ARH-77 cells, a B-lymphoblastoid cell line lacking significant expression of either syndecan-1 or heparanase, were transfected with the cDNA for heparanase. Following selection and confirmation that heparanase was stably expressed, the cells were analyzed by gene array and flow cytometry for syndecan-1 expression. Results show that expression of heparanase stimulates initiation of syndecan-1 transcription and expression on the cell surface. Karyotyping and analysis with a series of phenotypic markers for B cells show that the transfected ARH-77 cells maintain their general phenotype when syndecan-1 is upregulated by heparanase. Together these findings indicate that in addition to its role in cleaving heparan sulfate chains, the expression of heparanase upregulates the expression and shedding of syndecan-1 in tumor cells. Thus, the promotion by heparanase of tumor growth, angiogenesis and metastasis may, at least in part, be due to its positive effects on syndecan-1 expression and shedding which are also known to promote tumor progression in myeloma. Inhibitors of heparanase now being tested clinically may thus have the dual effect of blocking heparanase enzyme activity and decreasing syndecan-1 expression, both which could negatively affect tumor growth and metastasis.

Heparanase promotes growth, angiogenesis and survival of primary breast tumors

Despite great strides toward diagnosis and therapy, breast cancer remains a most threatening disease in its incidence, morbidity and mortality; therefore, additional knowledge regarding the molecular mechanisms contributing to breast cancer progression, as well as new targets for drug discovery are highly needed. Heparanase is the predominant enzyme involved in cleavage of heparan sulfate, the main polysaccharide component of the extracellular matrix. Experimental and clinical data indicate that heparanase plays important roles in cancer metastasis and angiogenesis. In breast carcinoma patients, heparanase expression correlates with the metastatic potential of the tumor. The present study was undertaken to investigate the role of heparanase in local growth and angiogenesis of primary breast tumors. MCF-7 breast carcinoma cells were stable transfected with the human heparanase (H-hpa) cDNA, or empty vector (mock), and injected into the mammary pad of nude mice. MRI was applied to monitor progression of tumor growth and angiogenesis. We demonstrate that tumors produced by cells overexpressing heparanase grew faster and were 7-fold larger than tumors produced by mock transfected cells. This enhanced growth was accompanied by increased tumor vascularization and a higher degree of vessel maturation. Histological examination ascribed the differences in tumor growth to heparanase-stimulated cell proliferation and survival. In-vitro experiments reinforced heparanase role as a survival factor under stress conditions. Moreover, H-hpa tumor cells infiltrate into the adjacent stroma, promoting formation of highly vascularized fibrous bands. Our results emphasize the significance and clarify the involvement of heparanase in primary breast cancer progression by generating a supportive microenvironment that promotes tumor growth, angiogenesis and survival.

Expression of heparanase and its clinical significance in human non-small cell lung cancer

Objective: To explore the expression and significance of heparanase in non-small cell lung cancer (NSCLC) regarding prognosis and clinicopathological parameters. Methods: The expression of heparanase was assessed using immunohistochemistry staining and Western blot in 122 paraffin-embedded specimens and 38 freshly taken tissues. The relationship between heparanase expression and the clinicopathological factors was analyzed by Chi-square test, multivariate analysis and Kaplan-Meier method. Results: In the immunoreactive cells, staining was mainly located in cytoplasma and membrane. Human heparanase was highly expressed in lung cancer tissue (78.7%, 96/122) while negative in epithelia of normal lung tissues. The level of heparanase was remarkably higher in NSCLC than that in normal tissue (P=0.043). Expression of heparanase significantly correlated with TNM stage (P=0.025), lymphatic metastasis (P=0.002) and vascular invasion (P=0.0003). The patients with positive heparanase expression had a significantly shorter survival than those with negative heparanase expression (P=0.0006). In multivariate analysis, only p-TNM stage, lymphatic metastasis and vascular invasion could be considered as prognostic factors. Conclusion: Elevated level of heparanase in human non-small cell lung cancer tissues correlates with the TNM stage, invasion, metastasis and prognosis. However, heparanase expression is not an independent prognostic factor.

Expression of Heparanase by Primary Breast Tumors Promotes Bone Resorption in the Absence of Detectable Bone Metastases

Heparanase is an enzyme that cleaves heparan sulfate and through this activity promotes tumor growth, angiogenesis, invasion, and metastasis in several tumor types. In human breast cancer patients, heparanase expression is associated with sentinel lymph node metastases. However, the precise role of heparanase in the malignant progression of breast cancer is unknown. To examine this, a variant of MDA-MB-231 cells was transfected with the cDNA for human heparanase (HPSE cells) or with vector alone as a control (NEO cells). Transfection produced a 6-fold increase in heparanase activity in HPSE cells relative to NEO cells. When injected into the mammary fat pads of severe combined immunodeficient mice, the tumors formed by HPSE cells initially grow significantly faster than the tumors formed by NEO cells. The rapid growth is due in part to increased angiogenesis, as microvessel densities are substantially elevated in primary HPSE tumors compared with NEO tumors. Although metastases to bones are not detected, surprisingly vigorous bone resorption is stimulated in animals bearing tumors formed by the HPSE cells. These animals have high serum levels of the C-telopeptide derived from type I collagen as well as significant elevation of the active form of tartrate-resistant acid phosphatase (TRAP)-5b. In contrast, in animals having a high tumor burden of Neo cells, the serum levels of C-telopeptide and TRAP-5b never increase above the levels found before tumor injection. Consistent with these findings, histologic analysis for TRAP-expressing cells reveals extensive osteoclastogenesis in animals harboring HPSE tumors. In vitro osteoclastogenesis assays show that the osteoclastogenic activity of HPSE cell conditioned medium is significantly enhanced beyond that of NEO conditioned medium. This confirms that a soluble factor or factors that stimulate osteoclastogenesis are specifically produced when heparanase expression is elevated. These factors exert a distal effect resulting in resorption of bone and the accompanying enrichment of the bone microenvironment with growth-promoting factors that may nurture the growth of metastatic tumor cells. This novel role for heparanase as a promoter of osteolysis before tumor metastasis suggests that therapies designed to block heparanase function may disrupt the early progression of bone-homing tumors.

Identification and Characterization of Heparin/Heparan Sulfate Binding Domains of the Endoglycosidase Heparanase

The endo-beta-glucuronidase, heparanase, is an enzyme that cleaves heparan sulfate at specific intra-chain sites, yielding heparan sulfate fragments with appreciable size and biological activities. Heparanase activity has been traditionally correlated with cell invasion associated with cancer metastasis, angiogenesis, and inflammation. In addition, heparanase up-regulation has been documented in a variety of primary human tumors, correlating with increased vascular density and poor postoperative survival, suggesting that heparanase may be considered as a target for anticancer drugs. In an attempt to identify the protein motif that would serve as a target for the development of heparanase inhibitors, we looked for protein domains that mediate the interaction of heparanase with its heparan sulfate substrate. We have identified three potential heparin binding domains and provided evidence that one of these is mapped at the N terminus of the 50-kDa active heparanase subunit. A peptide corresponding to this region (Lys(158)-Asp(171)) physically associates with heparin and heparan sulfate. Moreover, the peptide inhibited heparanase enzymatic activity in a dose-responsive manner, presumably through competition with the heparan sulfate substrate. Furthermore, antibodies directed to this region inhibited heparanase activity, and a deletion construct lacking this domain exhibited no enzymatic activity. NMR titration experiments confirmed residues Lys(158)-Asn(162) as amino acids that firmly bound heparin. Deletion of a second heparin binding domain sequence (Gln(270)-Lys(280)) yielded an inactive enzyme that failed to interact with cell surface heparan sulfate and hence accumulated in the culture medium of transfected HEK 293 cells to exceptionally high levels. The two heparin/heparan sulfate recognition domains are potentially attractive targets for the development of heparanase inhibitors.

In vivo fragmentation of heparan sulfate by heparanase overexpression renders mice resistant to amyloid protein A amyloidosis

Amyloid diseases encompass >20 medical disorders that include amyloid protein A (AA) amyloidosis, Alzheimer's disease, and type 2 diabetes. A common feature of these conditions is the selective organ deposition of disease-specific fibrillar proteins, along with the sulfated glycosaminoglycan, heparan sulfate. We have generated transgenic mice that overexpress human heparanase and have tested their susceptibility to amyloid induction. Drastic shortening of heparan sulfate chains was observed in heparanase-overproducing organs, such as liver and kidney. These sites selectively escaped amyloid deposition on experimental induction of inflammation-associated AA amyloidosis, as verified by lack of material staining with Congo Red, as well as lack of associated polysaccharide, whereas the same tissues from control animals were heavily infiltrated with amyloid. By contrast, the spleens of transgenic mice that failed to significantly overexpress heparanase contained heparan sulfate chains similar in size to those of control spleen and remained susceptible to amyloid deposition. Our findings provide direct in vivo evidence that heparan sulfate is essential for the development of amyloid disease.

Site-directed Mutagenesis, Proteolytic Cleavage, and Activation of Human Proheparanase

Heparanase is an endo-beta-D-glucuronidase that degrades heparan sulfate in the extracellular matrix and cell surfaces. Human proheparanase is produced as a latent 65-kDa polypeptide undergoing processing at two potential proteolytic cleavage sites, located at Glu109-Ser110 (site 1) and Gln157-Lys158 (site 2). Cleavage of proheparanase yields 8- and 50-kDa subunits that heterodimerize to form the active enzyme. The fate of the linker segment (Ser110-Gln157) residing between the two subunits, the mode of processing, and the protease(s) engaged in proheparanase processing are currently unknown. We applied multiple site-directed mutagenesis and deletions to study the nature of the potential cleavage sites and amino acids essential for processing of proheparanase in transfected human choriocarcinoma cells devoid of endogenous heparanase but possessing the enzymatic machinery for proper processing and activation of the proenzyme. Although mutagenesis at site 1 and its flanking sequences failed to identify critical residues for proteolytic cleavage, processing at site 2 required a bulky hydrophobic amino acid at position 156 (i.e. P2 of the cleavage site). Substitution of Tyr156 by Ala or Glu, but not Val, resulted in cleavage at an upstream site in the linker segment, yielding an improperly processed inactive enzyme. Processing of the latent 65-kDa proheparanase in transfected Jar cells was inhibited by a cell-permeable inhibitor of cathepsin L. Moreover, recombinant 65-kDa proheparanase was processed and activated by cathepsin L in a cell-free system. Altogether, these results suggest that proheparanase processing at site 2 is brought about by cathepsin L-like proteases. The involvement of other members of the cathepsin family with specificity to bulky hydrophobic residues cannot be excluded. Our results and a three-dimensional model of the enzyme are expected to accelerate the design of inhibitory molecules capable of suppressing heparanase-mediated enhancement of tumor angiogenesis and metastasis.

Positive association of heparanase expression with tumor invasion and lymphatic metastasis in gastric carcinoma

Tumor invasion and metastasis are the most common causes of death in gastric carcinoma. Human heparanase influences tumor invasiveness and angiogenesis. Analysis of its expression in gastric carcinoma has been hindered by our inability to procure pure cancer cells from heterogeneous tissue. In the present study, we analyzed heparanase expression in human primary and metastatic gastric carcinoma cells as well as in paired normal gastric epithelial cells by laser capture microdissection coupled with reverse transcription-polymerase chain reaction (RT-PCR). Tumor tissues, metastatic lymph nodes, and apparently uninvolved normal gastric tissues were collected from 30 patients who had undergone gastrectomy with radical lymph node dissection for gastric carcinoma without preoperative treatment. Bulk tissues and laser capture microdissected cell groups were separately subjected to RT-PCR analysis with heparanase-specific primers. For bulk tissues, heparanase-specific transcripts were detectable in all primary tumor tissues, metastatic lymph nodes, and almost all matching normal tissues. RT-PCR analysis after laser capture microdissection showed no detectable heparanase expression in matching normal epithelial cell groups. Of the laser capture microdissected primary gastric carcinoma cells, 47% (14/30) were heparanase positive. Expression was closely associated with greater tumor invasiveness, including Borrmann gross type and depth of wall infiltration. For metastatic cell groups dissected from lymph nodes, 95% showed clear heparanase expression. Furthermore, the extent of lymphatic spread was directly correlated to heparanase expression at the primary site. In conclusion, laser capture microdissection coupled with RT-PCR is a reliable approach for molecular analysis of heparanase expression in gastric carcinoma. Heparanase may facilitate invasion and metastasis of gastric carcinoma cells.

Clinicopathological significance of heparanase and basic fibroblast growth factor expression in human esophageal cancer

Human heparanase is an endo-D-glucuronidase that degrades heparan sulfate/heparin and has been implicated in a variety of biological processes. The objective was to investigate the expression of heparanase (Hps) and basic fibroblast growth factor (bFGF) and their relationship to neoangiogenesis and metastasis of human esophageal carcinoma. Seventy-nine patients who had undergone esophageal resection for esophageal carcinoma without preoperative treatment were included in the present study. Immunohistochemistry was used to study the expression of Hps, bFGF and microvessel density (MVD) in 79 cases of esophageal carcinoma. bFGF and Hps were quantitatively detected with immunohistochemistry in 79 cases of human esophageal carcinoma and 19 cases of adjacent normal human esophageal carcinoma. Cd34 was used to explore the MVD as a marker of endothelial cells. Hps and bFGF expression in tumor tissue, being remarkably higher than that in normal esophageal tissue, were significantly correlated with clinicopathological features (depth of invasion, lymph-node metastasis and TNM stage) and MVD. The results of this study suggest that the coexpression of Hps and bFGF plays a key role in angiogenesis, invasion and metastasis of esophageal carcinoma. Hps and bFGF may serve as a predictor of progression in esophageal carcinoma. The expression of heparanase in esophageal carcinoma enhances growth, invasion, and angiogenesis of the tumor, and bFGF seems to be a potent antigenic factor for esophageal carcinoma.

Clinical significance of heparanase and basic fibroblast growth factor expression in human non-small cell lung cancer

To assess protein and mRNA expression levels of heparanase and basic fibroblast growth factor (bFGF) genes in human non-small cell lung cancer (NSCLC) and their roles in tumor invasion, metastasis and prognosis. A total of 115 paraffin-embedded and 45 fresh-frozen tissue specimens of NSCLC were studied by immunohistochemistry, Western Blot and in situ hybridization to evaluate the protein and mRNA expression status of heparanase and bFGF genes. The data was analyzed by SPSS statistical software. Both human heparanase and bFGF were highly expressed in NSCLC cells, in contrast to none or a low expression in normal lung tissue. Expression of heparanase also showed a significantly higher than that in the normal tissue by Western blot (P = 0.041). Immunohistochemistry showed that heparanase expression was both cytoplasmic and membranous. The agreement between heparanase and bFGF was significant. A significant correlation was found between the expression of either protein and TNM stage, vascular invasion, lymphatic metastasis and microvascular density (MVD). Co-expression of the two proteins demonstrated an even higher correlation with the tumor stage and MVD. In addition, expression of bFGF correlated with tumor cell differentiation. Data of a multivariate analysis indicated that tumor cell differentiation, vascular invasion, lymphatic metastasis and expression of bFGF were identified as significant prognostic parameters. Both heparanase and bFGF may play important roles in tumor angiogenesis, metastasis, and prognosis of NSCLC.

Heparanase mRNA Gene and Protein Expression in Oral Cancer Development and Progression

The human heparanase gene, an endo-beta-D-glucuronidase, has recently been cloned. It functions as an extracellular degradative enzyme that cleaves heparan sulfate proteoglycans, and acts as a critical modulator of tumor metastasis and angiogenesis. Heparanase RNA probe and monoclonal anti-heparanase antibodies were used to examine the expression of heparanase mRNA gene and protein in epithelial dysplasia, carcinoma-in-situ and/or microinvasive carcinoma and oral squamous cell carcinoma. Strong signal and expression of both gene and protein were detected in epithelium progressing from dysplasia to invasive carcinoma. Carcinomatous cells at the tumor invasive front showed the highest levels of heparanase mRNA gene and protein. These results suggest that heparanase plays an important role during oral cancer development and progression, and this may have both prognostic and therapeutic implications.

Heparanase gene silencing, tumor invasiveness, angiogenesis, and metastasis.

Heparanase is an endoglycosidase that degrades heparan sulfate, the main polysaccharide constituent of the extracellular matrix and basement membrane. Expression of the heparanase gene is associated with the invasive, angiogenic, and metastatic potential of diverse malignant tumors and cell lines. We used gene-silencing strategies to evaluate the role of heparanase in malignancy and to explore the therapeutic potential of its specific targeting. We designed plasmid vectors to express hammerhead ribozymes or small interfering RNAs (siRNAs) directed against the human or mouse heparanase mRNAs. Human breast carcinoma (MDA-MB-435) and mouse lymphoma (Eb) and melanoma (B16-BL6) tumor cell lines, which have naturally high levels of endogenous heparanase or have been genetically engineered to overexpress heparanase, were transfected with anti-heparanase ribozyme or siRNA. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and measurements of enzymatic activity were used to confirm the efficient silencing of heparanase gene expression. Cells transfected with the anti-heparanase ribozyme and siRNA vectors were tested for invasiveness in vitro and metastatic dissemination in animal models of experimental and spontaneous metastasis. Compared with cells transfected with control constructs, cells transfected with the anti-heparanase ribozyme or siRNA vectors had profoundly reduced invasion and adhesion in vitro, regardless of cell type, and expressed less heparanase. In vivo, tumors produced by cells transfected with the anti-heparanase ribozyme and siRNA vectors were less vascularized and less metastatic than tumors produced by cells transfected with the control vectors. Mice injected with cells transfected with the anti-heparanase ribozyme and siRNA vectors lived longer than mice injected with control cells. The association of reduced levels of heparanase and altered tumorigenic properties in cells with anti-heparanase ribozyme- or siRNA-mediated gene-silencing vectors suggests that heparanase is important in cancer progression. Heparanase gene silencing has potential use as a target for anticancer drug development.

Increased expression of heparanase mRNA is correlated with metastasis of gastric carcinoma and is probably correlated with c-met protein

To explore the expression of human heparanase mRNA and its relationship with the clinicopathological characteristics and angiogenesis and its possible mechanism in gastric carcinomas. Expression of heparanase mRNA in gastric cancerous tissues and its corresponding adjacent tissues were detected by RT-PCR in forty-seven patients. Expression of c-met protein and microvessel density (MVD) were examined by immunohistochemcal staining. Expression of heparanse mRNA was significantly higher in gastric cancerous tissues than in its adjacent tissues (74.5% vs. 19.1%, P < 0.01). MVD was significantly higher in heparanase positive group than its counterpart (51 +/- 15 vs. 31 +/- 10, P < 0.01). Expression of heparanase mRNA was correlated with expression of c-met (r = 0.714, P < 0.01). The positive rate of heparanse expression in gastric carcinomas were correlated with tumor diameter, invasive depth, regional lymph nodes metastasis and TNM stage, whereas there were no relation with gender, age, tumor differentiation and remote metastasis of the patient. Increased heparanase expression contributes to metastasis and angiogenesis in gastric cancer. Heparanase mRNA expression correlates with poor clinicopathologic characteristics in its prognosis. C-met is correlated with heparanase expression.

Human heparanase nuclear localization and enzymatic activity

In previous studies, we have demonstrated that human heparanase (endo-β-D-glucuronidase) is localized primarily in a perinuclear pattern within lysosomes and late endosomes, and occasionally may be surface associated and secreted. The presence of two potential nuclear localization sequences in human heparanase, led us to investigate heparanase translocation into the nucleus and subsequent degradation of nuclear heparan sulfate. Applying cell fractionation, Western blot analysis, determination of heparanase activity and confocal microscopy, we identified heparanase within the nuclei of human glioma and breast carcinoma cells and estimated its amount to be about 7% of the cytosolic enzyme. Our results indicate that nuclear heparanase colocalizes with nuclear heparan sulfate and is enzymaticaly active. Moreover, following uptake of latent 65 kDa heparanase by cells that do not express the enzyme, an active 50 kDa heparanase was detected in the cell nucleus, capable of degrading both nuclear and extracellular matrix-derived heparan sulfate. Immunohistochemical examination of human squamous cell carcinoma specimens revealed a prominent granular staining of heparanase within the nuclei of the epithelial tumor cells vs no nuclear staining in the adjacent stromal cells. Taken together, it appears that heparanase is translocated into the cell nucleus where it may degrade the nuclear heparan sulfate and thereby affect nuclear functions that are thought to be regulated by heparan sulfate. Nuclear localization of heparanase suggests that the enzyme may fulfill nontraditional functions (ie, regulation of gene expression and signal transduction) apart of its well-documented involvement in cancer metastasis, angiogenesis and inflammation.