Adaptive evolution of heparanase in hypoxia-tolerant Spalax: gene cloning and identification of a unique splice variant

Heparan sulfate (HS) side chains of HS proteoglycans bind to and assemble extracellular matrix proteins and play important roles in cell-cell and cell-extracellular matrix interactions. HS chains bind a multitude of bioactive molecules and thereby function in the control of multiple normal and pathological processes. Enzymatic degradation of HS by heparanase, a mammalian endoglycosidase, affects the integrity and functional state of tissues and is involved in, among other processes, inflammation, angiogenesis, and cancer metastasis. Here, we report the cloning of heparanase from four Israeli species of the blind subterranean mole rat (Spalax ehrenbergi superspecies), 85% homologous to the human enzyme. Unlike its limited expression in human tissues, heparanase is highly expressed in diverse Spalax tissues. Moreover, we have identified a unique splice variant of the Spalax enzyme lacking 16 aa encoded by exon 7. This deletion resulted in a major defect in trafficking and processing of the heparanase protein, leading to a loss of its enzymatic activity. Interspecies variation was noted in the sequence and in the expression of the splice variant of the heparanase gene in blind mole rats living under different ecogeographical stresses, indicating a possible role in adaptation to stress in Spalax evolution.     

Heparanase-neutralizing antibodies attenuate lymphoma tumor growth and metastasis

Heparanase is an endoglycosidase that cleaves heparan sulfate side chains of proteoglycans, resulting in disassembly of the extracellular matrix underlying endothelial and epithelial cells and associating with enhanced cell invasion and metastasis. Heparanase expression is induced in carcinomas and sarcomas, often associating with enhanced tumor metastasis and poor prognosis. In contrast, the function of heparanase in hematological malignancies (except myeloma) was not investigated in depth. Here, we provide evidence that heparanase is expressed by human follicular and diffused non-Hodgkin''s B-lymphomas, and that heparanase inhibitors restrain the growth of tumor xenografts produced by lymphoma cell lines. Furthermore, we describe, for the first time to our knowledge, the development and characterization of heparanase-neutralizing monoclonal antibodies that inhibit cell invasion and tumor metastasis, the hallmark of heparanase activity. Using luciferase-labeled Raji lymphoma cells, we show that the heparanase-neutralizing monoclonal antibodies profoundly inhibit tumor load in the mouse bones, associating with reduced cell proliferation and angiogenesis. Notably, we found that Raji cells lack intrinsic heparanase activity, but tumor xenografts produced by this cell line exhibit typical heparanase activity, likely contributed by host cells composing the tumor microenvironment. Thus, the neutralizing monoclonal antibodies attenuate lymphoma growth by targeting heparanase in the tumor microenvironment.Heparanase is an endo-β-d-glucuronidase capable of cleaving heparan sulfate (HS) side chains at a limited number of sites, releasing saccharide products with appreciable size (4–7 kDa) and biological potency. Enzymatic degradation of HS leads to disassembly of the extracellular matrix (ECM) and correlates with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of HS cleavage and remodeling of the ECM and basement membrane underlying epithelial and endothelial cells (1, 2). Heparanase expression is induced in human cancer, most often associating with reduced patients’ survival postoperation, increased tumor metastasis, and higher vessel density (3–5). In addition, heparanase up-regulation is associated with tumors larger in size (3, 5). Likewise, heparanase over-expression enhanced (6, 7), whereas local delivery of anti-heparanase siRNA inhibited (8), the growth of tumor xenografts. These results imply that heparanase function is not limited to tumor metastasis but is engaged in progression of the primary lesion, thus critically supporting the intimate involvement of heparanase in tumor progression and encouraging the development of heparanase inhibitors as anticancer therapeutics (9–12). As a consequence, heparanase inhibitors are currently evaluated in phase 1 clinical trials (13).Heparanase activity is similarly implicated in the progression of multiple myeloma (14–16), but its significance in other hematologic malignancies has not yet been characterized. Lymphomas are a heterogeneous group of cancers that arise from developing lymphocytes and produce tumors predominantly in lymphoid structures (i.e., bone marrow), but also in extranodal tissues. Collectively, lymphomas constitute the fifth most common cancer in North America, with more than 90% of the patients being affected by lymphomas of B-cell origin (17). Despite overall improvements in outcomes of lymphoma, ∼30–40% of patients have disease that is either refractory or relapses after standard therapy (18). Therefore, a better understanding of the molecular pathobiology of lymphomas is needed for the development of new therapeutic approaches. Here, we provide evidence that heparanase is expressed by B-lymphomas and that heparanase inhibitors restrain tumor growth. Furthermore, we describe the development of novel heparanase-neutralizing monoclonal antibodies (mAbs) that attenuate lymphoma growth by targeting heparanase in the tumor microenvironment.     

Heparanase, tissue factor, and cancer

Heparanase is an endo-beta- D-glucuronidase that is capable of cleaving heparan sulfate side chains of heparan sulfate proteoglycans on cell surfaces and the extracellular matrix, activity that is strongly implicated in tumor metastasis and angiogenesis. Evidence was provided that heparanase overexpression in human leukemia, glioma, and breast carcinoma cells results in a marked increase in tissue factor (TF) levels. Likewise, TF was induced by exogenous addition of recombinant heparanase to tumor cells and primary endothelial cells, induction that was mediated by p38 phosphorylation and correlated with enhanced procoagulant activity. TF induction was further confirmed in heparanase-overexpressing transgenic mice and correlated with heparanase expression levels in leukemia patients. Heparanase was also found to be involved in the regulation of tissue factor pathway inhibitor (TFPI). It was shown that heparanase overexpression or exogenous addition induces two- to threefold increase of TFPI expression. Similarly, heparanase stimulated accumulation of TFPI in the cell culture medium. Extracellular accumulation exceeded, however, the observed increase in TFPI at the protein level and appeared to be independent of heparan sulfate and heparanase enzymatic activity. Instead, a physical interaction between heparanase and TFPI was demonstrated, suggesting a mechanism by which secreted heparanase interacts with TFPI on the cell surface, leading to dissociation of TFPI from the cell membrane and increased coagulation activity, thus further supporting the local prothrombotic function of heparanase. As heparins are strong inhibitors of heparanase, in view of the effect of heparanase on TF/TFPI pathway, the role of heparins' anticoagulant activity may potentially be expanded.     

Intron self-complementarity enforces exon inclusion in a yeast pre-mRNA

Skipping of internal exons during removal of introns from pre-mRNA must be avoided for proper expression of most eukaryotic genes. Despite significant understanding of the mechanics of intron removal, mechanisms that ensure inclusion of internal exons in multi-intron pre-mRNAs remain mysterious. Using a natural two-intron yeast gene, we have identified distinct RNA–RNA complementarities within each intron that prevent exon skipping and ensure inclusion of internal exons. We show that these complementarities are positioned to act as intron identity elements, bringing together only the appropriate 5′ splice sites and branchpoints. Destroying either intron self-complementarity allows exon skipping to occur, and restoring the complementarity using compensatory mutations rescues exon inclusion, indicating that the elements act through formation of RNA secondary structure. Introducing new pairing potential between regions near the 5′ splice site of intron 1 and the branchpoint of intron 2 dramatically enhances exon skipping. Similar elements identified in single intron yeast genes contribute to splicing efficiency. Our results illustrate how intron secondary structure serves to coordinate splice site pairing and enforce exon inclusion. We suggest that similar elements in vertebrate genes could assist in the splicing of very large introns and in the evolution of alternative splicing.     

PI-88 and novel heparan sulfate mimetics inhibit angiogenesis

The heparan sulfate (HS) mimetic PI-88 is a promising inhibitor of tumor growth and metastasis expected to commence phase III clinical evaluation in 2007 as an adjuvant therapy for postresection hepatocellular carcinoma. Its anticancer properties are attributed to inhibition of angiogenesis via antagonism of the interactions of angiogenic growth factors and their receptors with HS. It is also a potent inhibitor of heparanase, an enzyme that plays a key role in both metastasis and angiogenesis. A series of PI-88 analogs have been prepared with enhanced chemical and biological properties. The new compounds consist of single, defined oligosaccharides with specific modifications designed to improve their pharmacokinetic properties. These analogs all inhibit heparanase and bind to the angiogenic fibroblast growth factor 1 (FGF-1), FGF-2, and vascular endothelial growth factor with similar affinity to PI-88. However, compared with PI-88, some of the newly designed compounds are more potent inhibitors of growth factor-induced endothelial cell proliferation and of endothelial tube formation on Matrigel. Representative compounds were also tested for antiangiogenic activity in vivo and were found to reduce significantly blood vessel formation. Moreover, the pharmacokinetic profile of several analogs was also improved, as evidenced primarily by lower clearance in comparison with PI-88. The current data support the development of HS mimetics as potent antiangiogenic anticancer agents.     

Heparanase and hepatocellular carcinoma: Promoter or inhibitor?

Heparan sulphate proteoglycans (HSPGs) consist of a core protein and several heparan sulphate (HS) side chains covalently linked. HS also binds a great deal of growth factors, chemokines, cytokines and enzymes to the extracellular matrix and cell surface. Heparanase can specially cleave HS side chains from HSPGs. There are a lot of conflicting reports about the role of heparanase in hepatocellular carcinoma (HCC). Heparanase is involved in hepatitis B virus infection and hepatitis C virus infection, the act...     

Properties and function of heparanase in cancer metastasis and angiogenesis

Cleavage of heparan sulfate proteoglycans 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 sulfate (HS) at specific intrachain sites, is synthesized as a latent approximately 65 kDa protein that is processed at the N-terminus into a highly active approximately 50 kDa form. The heparanase enzyme is preferentially expressed in human tumors and its overexpression in low-metastatic tumor cells confers a highly invasive phenotype in experimental animals. Heparanase also releases angiogenic factors and accessory fragments of HS from the tumor microenvironment and induces an angiogenic response in vivo. These effects were best demonstrated when the enzyme was secreted and/or expressed on the cell surface. Heparanase may thus facilitate tumor cell invasion, vascularization and survival, all critical events in cancer progression. These observations, the anti-cancerous 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.     

Heparanase is highly expressed and regulates proliferation in GH-secreting pituitary tumor cells

Pituitary tumorigenesis involves remodeling of the extracellular matrix (ECM). Heparanase, an endoglycosidase capable of degrading heparan sulfate, a major polysaccharide constituent of the ECM, is implicated in diverse processes associated with ECM remodeling, such as morphogenesis, angiogenesis, and tumor invasion. The aim of this study was to investigate the possible role of heparanase in pituitary tumorigenesis. Human normal pituitaries and pituitary tumors were examined for heparanase mRNA and protein expression using real-time PCR and immunohistochemistry, respectively. Cell proliferation was assessed by colony formation after heparanase overexpression in GH3 and MtT/S cells. Cell viability and cell cycle progression were evaluated after heparanase gene silencing. Higher heparanase mRNA and protein expression was noted in GH tumors as compared with normal pituitaries. Heparanase overexpression in GH3 and MtT/S cells resulted in a 2- to 3-fold increase in colony number, compared with control cells. Cell viability decreased by 50% after heparanase gene silencing due to induced apoptosis reflected by increased fraction of cleaved poly-ADP-ribose polymerase and sub-G1 events. Notably, exogenously added heparanase enhanced epidermal growth factor receptor, Src, Akt, ERK, and p38 phosphorylation in pituitary tumor cells. Our results indicate that heparanase enhances pituitary cell viability and proliferation and may thus contribute to pituitary tumor development and progression.     

Dramatic regulation of heparanase activity and angiogenesis gene expression in synovium from patients with rheumatoid arthritis

OBJECTIVE: Although heparanase is recognized as a proangiogenic factor, the involvement of heparanase in rheumatoid arthritis (RA) is unclear. In this study, we assessed heparanase activity in synovial fluid (SF) and synovial tissue (ST) from patients with RA or osteoarthritis (OA), and analyzed the expression of angiogenic pathway-focused genes in ST from RA and OA patients. METHODS: SF and ST were obtained from the knees of patients with either RA or OA and from asymptomatic donors with no documented history of degenerative or inflammatory joint diseases. Heparanase activity was determined by an enzymatic assay using a radiolabeled substrate, and the presence of heparanase in ST was demonstrated by Western blotting. The expression of angiogenesis genes, including heparanase, in ST was analyzed by real-time quantitative polymerase chain reaction. RESULTS: Heparanase activity was dramatically higher (>100-fold) in SF and ST from RA patients than in SF and ST from OA patients and asymptomatic donors. Active heparanase enzyme was detected and heparanase messenger RNA was up-regulated in ST from RA patients. We also found that angiogenesis gene expression was significantly regulated in RA synovium, and was correlated with heparanase activity. CONCLUSION: These findings are novel and contribute to our understanding of joint destruction in RA, suggesting that heparanase may be a reliable prognostic factor for RA progression and an attractive target for the treatment of RA.     

Heparanase enhances the generation of activated factor X in the presence of tissue factor and activated factor VII

Background

Heparanase is an endo-β-D-glucuronidase dominantly involved in tumor metastasis and angiogenesis. Recently, we demonstrated that heparanase is involved in the regulation of the hemostatic system. Our hypothesis was that heparanase is directly involved in activation of the coagulation cascade.

Design and Methods

Activated factor X and thrombin were studied using chromogenic assays, immunoblotting and thromboelastography. Heparanase levels were measured by enzyme-linked immunosorbent assay. A potential direct interaction between tissue factor and heparanase was studied by co-immunoprecipitation and far-western assays.

Results

Interestingly, addition of heparanase to tissue factor and activated factor VII resulted in a 3- to 4-fold increase in activation of the coagulation cascade as shown by increased activated factor X and thrombin production. Culture medium of human embryonic kidney 293 cells over-expressing heparanase and its derivatives increased activated factor X levels in a non-enzymatic manner. When heparanase was added to pooled normal plasma, a 7- to 8-fold increase in activated factor X level was observed. Subsequently, we searched for clinical data supporting this newly identified role of heparanase. Plasma samples from 35 patients with acute leukemia at presentation and 20 healthy donors were studied for heparanase and activated factor X levels. A strong positive correlation was found between plasma heparanase and activated factor X levels (r=0.735, P=0.001). Unfractionated heparin and an inhibitor of activated factor X abolished the effect of heparanase, while tissue factor pathway inhibitor and tissue factor pathway inhibitor-2 only attenuated the procoagulant effect. Using co-immunoprecipitation and far-western analyses it was shown that heparanase interacts directly with tissue factor.

Conclusions

Overall, our results support the notion that heparanase is a potential modulator of blood hemostasis, and suggest a novel mechanism by which heparanase increases the generation of activated factor X in the presence of tissue factor and activated factor VII.     

LHR splicing variants and gene expression in the marmoset monkey

In the marmoset monkey, the LHR type II, lacking exon 10, is the native receptor type. We characterised the LHR splicing pattern in marmoset testes and the adrenals during puberty and in pre- and postpubertal ovaries and quantified mRNA LHR expression in the testis. We detected 11 LHR splicing variants expressed at similar levels and generated by exon skipping and/or usage of cryptic splice sites. No preferred splicing variant during pubertal maturation was observed in both sexes. Testicular and adrenal LHR expression levels did not significantly change with age. However, a significant increase during pubertal maturation for the serum testosterone/LHR ratio indicated that testosterone secretion increases in the presence of constant LHR mRNA expression levels. We conclude that LHR splicing in the marmoset displays a homogenous pattern and that the main function of the LHR is established in the testis, reaching its highest efficiency during pubertal maturation.     

Degranulating mast cells secrete an endoglycosidase that degrades heparan sulfate in subendothelial extracellular matrix.

Mast cells are widely distributed in perivascular connective tissues, especially in areas of active tumor growth and vascular reactivity. Incubation of metabolically [35S]O4 = -labeled subendothelial extracellular matrix (ECM) with lysates of bone marrow-derived mouse mast cells (BMMC) resulted in extensive degradation of heparan sulfate (HS) into fragments 5 to 6 times smaller than intact HS side chains. A much lower activity (seven- to eightfold) was expressed by intact BMMC incubated in contact with the ECM. These fragments were not produced in the presence of heparin, were sensitive to deamination with nitrous acid, and resistant to further degradation with papain or chondroitinase ABC. These results indicate that an endoglycosidase (heparanase) is involved in BMMC-mediated degradation of HS in the subendothelial ECM. Heparanase activity was not detected in medium conditioned by cultured BMMC, or in lysates of Ableson transformed BMMC and rat basophilic leukemic (RBL) cells. Both heparanase and beta-hexosaminidase, a mast cell granule enzyme, were released on degranulation of BMMC induced by the calcium ionophore A23187, or by exposure to IgE-Ag, suggesting that heparanase is localized in the cell granules. Under these conditions, less than 5% of the cellular content of lactate dehydrogenase were released. Degradation of the ECM-HS by the mast cell heparanase and the associated release of HS-bound endothelial cell growth factors that are stored in ECM (Vlodavsky et al, Proc Natl Acad Sci USA 84:2292, 1987; Bashkin et al, Biochemistry 28:1737, 1989) may play a role in the proposed mast cell-mediated stimulation of neovascularization.     

Heparanase expression correlates with malignant potential in human colon cancer

Purpose Heparanase cleaves carbohydrate chains of heparan sulphate proteoglycans and is an important component of the extracellular matrix. This study was designed to determine the relation between heparanase expression and prognosis of patients with colon cancer.Methods The study included 54 patients (35 males and 19 females) who underwent colorectal resection for colorectal cancer between January 1992 and December 1994. Expression of heparanase protein and mRNA were determined and correlated with various clinicopathological parameters. In vitro studies were also performed to examine tumor invasion and to test the effects of heparanase inhibition, and in vivo studies were performed to examine tumor metastasis and prognosis.Results Heparanase expression was detected in the invasion front of the tumor in 37 of 54 (69%) colon cancer samples, whereas 17 of 54 (31%) tumors were negative. Expression of heparanase was significantly more frequent in tumors of higher TNM stage (P=0.0481), higher Dukes stage (P=0.0411), higher vascular infiltration (P=0.0146), and higher lymph vessel infiltration (P=0.0010). Heparanase expression in colon cancers correlated significantly with poor survival (P=0.0361). Heparanase-transfected colon cancer cells exhibited significant invasion compared with control-transfected colon cancer cells (P=0.001), and the peritoneal dissemination model also showed the malignant potential of heparanase-transfected cells, as assayed by number of nodules (P=0.017) and survival (P=0.0062). Inhibition of heparanase significantly reduced the invasive capacity of cancer cells (P=0.003).Conclusions Heparanase is a marker for poor prognosis of patients with colon cancer and could be a suitable target for antitumor therapy in colon cancer.     

An exon splice enhancer mutation causes autosomal dominant GH deficiency

Familial isolated GH deficiency type II (IGHD II) is caused, in some cases, by heterogeneous IVS3 mutations that affect GH mRNA splicing. We report here our finding an A-->G transition of the fifth base of exon 3 (E3+ 5 A-->G) in affected individuals from an IGHD II family. This mutation disrupts a (GAA)(n) exon splice enhancer (ESE) motif immediately following the weak IVS2 3' splice site. The mutation also destroys an MboII site used to demonstrate heterozygosity in all affected family members. To determine the effect of ESE mutations on GH mRNA processing, GH(3) cells were transfected with expression constructs containing the normal ESE, +5 A-->G, or other ESE mutations, and cDNAs derived from the resulting GH mRNAs were sequenced. All ESE mutations studied reduced activation of the IVS2 3' splice site and caused either partial E3 skipping, due to activation of an E3+ 45 cryptic 3' splice site, or complete E3 skipping. Partial or complete E3 skipping led to loss of the codons for amino acids 32-46 or 32-71, respectively, of the mature GH protein. Our data indicate that the E3+ 5 A-->G mutation causes IGHD II because it perturbs an ESE required for GH splicing.