Orit Goldshmidt

Cell surface expression and secretion of heparanase markedly promote tumor angiogenesis and metastasis

The present study emphasizes the importance of cell surface expression and secretion of heparanase (endo-β-d-glucuronidase) in tumor angiogenesis and metastasis. For this purpose, nonmetastatic Eb mouse lymphoma cells were transfected with the predominantly intracellular human heparanase or with a readily secreted chimeric construct composed of the human enzyme and the chicken heparanase signal peptide. Eb cells overexpressing the secreted heparanase invaded a reconstituted basement membrane to a much higher extent than cells overexpressing the intracellular enzyme. Cell invasion was inhibited in the presence of laminaran sulfate, a potent inhibitor of heparanase activity and experimental metastasis. The increased invasiveness in vitro was reflected in vivo by rapid and massive liver colonization and accelerated mortality. In fact, mice inoculated with cells expressing the secreted enzyme succumb because of liver metastasis and dysfunction, as early as 10 days after s.c. inoculation of the cells, when their tumor burden did not exceed 1% of body weight. Cell surface localization and secretion of heparanase markedly stimulated tumor angiogenesis, as demonstrated by a 4–6-fold increase in vessel density and functionality evaluated by MRI of tumors produced by cells expressing the secreted vs. the nonsecreted heparanase, consistent with actual counting of blood vessels. Altogether, our results indicate that the potent proangoigenic and prometastatic properties of heparanase are tightly regulated by its cellular localization and secretion. The increased potency of the secreted enzyme makes it a promising target for anticancer drug development.

Heparanase mediates cell adhesion independent of its enzymatic activity

Heparanase is an endo‐β‐D‐glucuronidase that cleaves heparan sulfate and is implicated in diverse physiological and pathological processes. In this study we report on a novel direct involvement of heparanase in cell adhesion. We demonstrate that expression of heparanase in nonadherent lymphoma cells induces early stages of cell adhesion, provided that the enzyme is expressed on the cell surface. Heparanasemediated cell adhesion to extracellular matrix (ECM) results in integrin‐dependent cell spreading, tyrosine phosphorylation of paxillin, and reorganization of the actin cytoskeleton. The surface‐bound enzyme also augments cell invasion through a reconstituted basement membrane. Cell adhesion was augmented by cell surface heparanase regardless of whether the cells were transfected with active or point mutated inactive enzyme, indicating that heparanase functions as an adhesion molecule independent of its endoglycosidase activity. The combined feature of heparanase as an ECM‐degrading enzyme and a cell adhesion molecule emphasizes its significance in processes involving cell adhesion, migration, and invasion, including embryonic development, neovascularization, and cancer metastasis.— Goldshmidt, O., Zcharia, E., Cohen, M., Aingorn, H., Cohen, I., Nadav, L., Katz, B.‐Z., Geiger, B., Vlodavsky, I. Heparanase mediates cell adhesion independent of its enzymatic activity. FASEB J. 17, 1015–1025 (2003)

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.

Molecular properties and involvement of heparanase in cancer progression and normal development.

Heparan sulfate proteoglycans (HSPGs) play a key role in the self-assembly, insolubility and barrier properties of basement membranes and extracellular matrices. Hence, cleavage of heparan sulfate (HS) 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. Here, we describe the molecular properties, expression and function of a human heparanase, degrading HS at specific intrachain sites. The enzyme 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 mRNA and protein are preferentially expressed in metastatic cell lines and human tumor tissues. Overexpression of the heparanase cDNA in low-metastatic tumor cells conferred a high metastatic potential in experimental animals, resulting in an increased rate of mortality. The heparanase enzyme also releases ECM-resident angiogenic factors in vitro and its overexpression induces an angiogenic response in vivo. Heparanase may thus facilitate both tumor cell invasion and neovascularization, both critical steps in cancer progression. The enzyme is also involved in cell migration associated with inflammation and autoimmunity. The unexpected identification of a single predominant functional heparanase suggests that the enzyme is a promising target for drug development. In fact, treatment with heparanase inhibitors markedly reduces tumor growth, metastasis and autoimmune disorders in animal models. Studies are underway to elucidate the involvement of heparanase in normal processes such as implantation, embryonic development, morphogenesis, tissue repair, inflammation and HSPG turnover. Heparanase is the first functional mammalian HS-degrading enzyme that has been cloned, expressed and characterized. This may lead to identification and cloning of other glycosaminoglycan degrading enzymes, toward a better understanding of their involvement and significance in normal and pathological processes.

Heparanase expression during normal liver development and following partial hepatectomy

Heparan sulphate proteoglycans are major components of the liver extracellular matrix. Their cleavage by heparanase (endo‐β‐glucuronidase) may thus be involved in liver‐specific normal and pathological processes. Heparanase mRNA and protein were expressed during liver development but not in the mature healthy liver. A biphasic gain of heparanase expression, detected by immunostaining, western blotting, and real‐time RT‐PCR, was clearly noted following partial hepatectomy, peaking at 12 and 96–168 h and subsiding 2 weeks post‐surgery. Expression of heparan sulphate gradually increased throughout the regeneration process. Unlike heparanase, baseline levels of matrix metalloproteinase‐2 (MMP‐2) were detected in the intact liver, increasing up to 4 days following partial hepatectomy and subsiding at day 10. Bands matching MMP‐9 were absent prior to hepatectomy, but visible 2 h post‐hepatectomy. Thioacetamide‐induced liver fibrosis was associated with increased levels of MMP‐9 and MMP‐2, correlating with the severity of the disease. Elevated heparanase levels were noted in the early stages of fibrosis, with no further increase evident in rats exhibiting higher fibrotic grades. Taken together, these data suggest a role for heparanase during liver development and remodelling. Copyright

Association of hereditary hemorrhagic telangiectasia and hereditary nonpolyposis colorectal cancer in the same kindred.

Endoglin (CD105) is a proliferation‐associated protein that is strongly expressed in endothelial tissue and has a role in tumor angiogenesis. Mutations in endoglin are also linked to Hereditary Hemorrhagic Telangiectasia type 1 (HHT1), an autosomal dominant disease associated with aberrant angiogenesis. We report an unusual association of HHT1 and Hereditary Nonpolyposis Colorectal Cancer (HNPCC) in the same kindred. Genetic analysis indicates that these 2 syndromes are genetically unrelated and separately segregated within the family. The mutation in the endoglin gene leads to a truncated protein. The mutation in the mismatch repair gene MLH1 causes a splicing defect, giving synthesis to an unstable mRNA from this mutated allele. The potential protective role of an endoglin mutation in patients with HNPCC is discussed.