Neta Ilan

Heparanase upregulation by colonic epithelium in inflammatory bowel disease

Heparanase is an endo-β-D-glucuronidase capable of cleaving heparan sulfate (HS) side chains at a limited number of sites, yielding HS fragments of still appreciable size (∼5–7u2009kDa). Heparanase activity has long been detected in a number of cell types and tissues. Importantly, heparanase activity correlated with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of HS cleavage and remodeling of the extracellular matrix barrier. Similarly, heparanase activity was implicated in neovascularization, inflammation and autoimmunity, involving migration of vascular endothelial cells and activated cells of the immune system. The involvement of heparanase in inflammatory processes of the gastrointestinal tract has not been examined. Here, we utilized immunohistochemical analysis to investigate heparanase expression in acute and chronic inflammatory conditions. Heparanase expression was not detected in specimens derived from normal colon tissue. In contrast, strong heparanase staining was observed in Crohns disease and ulcerative colitis, but not in infectious colitis. Interestingly, heparanase staining was primarily observed in epithelial rather than immune cells. Importantly, un-fractionated as well as low molecular weight heparin (enoxaparin), which exhibit a strong inhibitory activity towards heparanase, have proven efficacious in ulcerative colitis and Crohns disease patients, suggesting that heparanase is actively involved in these pathologies and thus may be considered as a target for the development of anti-inflammatory therapies.

Heparanase expression in nasopharyngeal carcinoma inversely correlates with patient survival

Aim :u2002To determine the expression and prognostic significance of heparanase in nasopharyngeal carcinoma (NPC).

Full-Length Semaphorin-3C Is an Inhibitor of Tumor Lymphangiogenesis and Metastasis

Semaphorins play important regulatory roles in diverse processes such as axon guidance, angiogenesis, and immune responses. We find that semaphorin-3C (sema3C) induces the collapse of the cytoskeleton of lymphatic endothelial cells (LEC) in a neuropilin-2-, plexin-D1-, and plexin-A1-dependent manner, while most other semaphorins, including antiangiogenic semaphorins such as sema3A do not. Sema3C is cleaved, like other class-3 semaphorins, by furin-like pro-protein convertases (FPPC). Cleaved sema3C (p65-Sema3C) was unable to induce the collapse of the cytoskeleton of LEC. FPPC are strongly upregulated in tumor cells. In order to examine the effects of full-length sema3C on tumor progression, we therefore generated an active point mutated furin cleavage-resistant sema3C (FR-sema3C). FR-sema3C inhibited potently proliferation of LEC and to a lesser extent proliferation of human umbilical vein-derived endothelial cells. FR-sema3C also inhibited VEGF-C-induced phosphorylation of VEGFR-3, ERK1/2, and AKT. Expression of recombinant FR-sema3C in metastatic, triple-negative LM2-4 breast cancer cells did not affect their migration or proliferation in vitro. However, tumors derived from FR-sema3C-expressing LM2-4 cells implanted in mammary fat pads developed at a slower rate, contained a lower concentration of blood vessels and lymph vessels, and metastasized much less effectively to lymph nodes. Interestingly, p65-Sema3C, but not FR-sema3C, rendered A549 lung cancer cells resistant to serum deprivation, suggesting that previously reported protumorigenic activities of sema3C may be due to p65-Sema3C produced by tumor cells. Our observations suggest that FR-sema3C may be further developed into a novel antitumorigenic drug.

Heparanase regulates retention and proliferation of primitive Sca-1+/c-Kit+/Lin− cells via modulation of the bone marrow microenvironment

Heparanase is involved in tumor growth and metastasis. Because of its unique cleavage of heparan sulfate, which binds cytokines, chemokines and proteases, we hypothesized that heparanase is also involved in regulation of early stages of hematopoiesis. We report reduced numbers of maturing leukocytes but elevated levels of undifferentiated Sca-1(+)/c-Kit(+)/Lin(-) cells in the bone marrow (BM) of mice overexpressing heparanase (hpa-Tg). This resulted from increased proliferation and retention of the primitive cells in the BM microenvironment, manifested in increased SDF-1 turnover. Furthermore, heparanase overexpression in mice was accompanied by reduced protease activity of MMP-9, elastase, and cathepsin K, which regulate stem and progenitor cell mobilization. Moreover, increased retention of the progenitor cells also resulted from up-regulated levels of stem cell factor (SCF) in the BM, in particular in the stem cell-rich endosteum and endothelial regions. Increased SCF-induced adhesion of primitive Sca-1(+)/c-Kit(+)/Lin(-) cells to osteoblasts was also the result of elevation of the receptor c-Kit. Regulation of these phenomena is mediated by hyperphosphorylation of c-Myc in hematopoietic progenitors of hpa-Tg mice or after exogenous heparanase addition to wildtype BM cells in vitro. Altogether, our data suggest that heparanase modification of the BM microenvironment regulates the retention and proliferation of hematopoietic progenitor cells.

Heparanase enhances myeloma progression via CXCL10 downregulation

In order to explore the mechanism(s) underlying the pro-tumorigenic capacity of heparanase, we established an inducible Tet-on system. Heparanase expression was markedly increased following addition of doxycycline (Dox) to the culture medium of CAG human myeloma cells infected with the inducible heparanase gene construct, resulting in increased colony number and size in soft agar. Moreover, tumor xenografts produced by CAG-heparanase cells were markedly increased in mice supplemented with Dox in their drinking water compared with control mice maintained without Dox. Consistently, we found that heparanase induction is associated with decreased levels of CXCL10, suggesting that this chemokine exerts tumor-suppressor properties in myeloma. Indeed, recombinant CXCL10 attenuated the proliferation of CAG, U266 and RPMI-8266 myeloma cells. Similarly, CXCL10 attenuated the proliferation of human umbilical vein endothelial cells, implying that CXCL10 exhibits anti-angiogenic capacity. Strikingly, development of tumor xenografts produced by CAG-heparanase cells overexpressing CXCL10 was markedly reduced compared with control cells. Moreover, tumor growth was significantly attenuated in mice inoculated with human or mouse myeloma cells and treated with CXCL10–Ig fusion protein, indicating that CXCL10 functions as a potent anti-myeloma cytokine.

Heparanase up-regulation in tongue cancer: tissue and saliva analysis.

Heparanase up‐regulation has been correlated with reduced postoperative survival in various cancers.

Macrophage-Induced Lymphangiogenesis and Metastasis following Paclitaxel Chemotherapy Is Regulated by VEGFR3

Summary While chemotherapy strongly restricts or reverses tumor growth, the response of host tissue to therapy can counteract its anti-tumor activity by promoting tumor re-growth and/or metastases, thus limiting therapeutic efficacy. Here, we show that vascular endothelial growth factor receptor 3 (VEGFR3)-expressing macrophages infiltrating chemotherapy-treated tumors play a significant role in metastasis. They do so in part by inducing lymphangiogenesis as a result of cathepsin release, leading to VEGF-C upregulation by heparanase. We found that macrophages from chemotherapy-treated mice are sufficient to trigger lymphatic vessel activity and structure in naive tumors in a VEGFR3-dependent manner. Blocking VEGF-C/VEGFR3 axis inhibits the activity of chemotherapy-educated macrophages, leading to reduced lymphangiogenesis in treated tumors. Overall, our results suggest that disrupting the VEGF-C/VEGFR3 axis not only directly inhibits lymphangiogenesis but also blocks the pro-metastatic activity of macrophages in chemotherapy-treated mice.

Processing of heparanase is mediated by syndecan-1 cytoplasmic domain and involves syntenin and α-actinin.

Heparanase activity plays a decisive role in cell dissemination associated with cancer metastasis. Cellular uptake of heparanase is considered a pre-requisite for the delivery of latent 65-kDa heparanase to lysosomes and its subsequent proteolytic processing and activation into 8- and 50-kDa protein subunits by cathepsin L. Heparan sulfate proteoglycans, and particularly syndecan, are instrumental for heparanase uptake and activation, through a process that has been shown to occur independent of rafts. Nevertheless, the molecular mechanism underlying syndecan-mediated internalization outside of rafts is unclear. Here, we examined the role of syndecan-1 cytoplasmic domain in heparanase processing, utilizing deletion constructs lacking the entire cytoplasmic domain (Delta), the conserved (C1 or C2), or variable (V) regions. Heparanase processing was markedly increased following syndecan-1 over-expression; in contrast, heparanase was retained at the cell membrane and its processing was impaired in cells over-expressing syndecan-1 deleted for the entire cytoplasmic tail. We have next revealed that conserved domain 2 (C2) and variable (V) regions of syndecan-1 cytoplasmic tail mediate heparanase processing. Furthermore, we found that syntenin, known to interact with syndecan C2 domain, and α actinin are essential for heparanase processing.

Plasma heparanase as a significant marker of treatment response in children with Hodgkin lymphoma: pilot study.

Introduction: The aim of this pilot study was to determine heparanase plasma levels (HP) at diagnosis and at restaging in children diagnosed with Hodgkin lymphoma and to investigate whether this parameter provides prognostic information for response to treatment after induction therapy. Patients and Methods: HP levels of 19 pediatric patients (mean age: 10.3 years (y) (range, 4–18 y), 9 girls, 10 boys) with Hodgkin lymphoma were assayed at diagnosis and at restaging. HP levels were determined using an ELISA anti-human heparanase immunoassay kit. According to diagnosis, CAT scan and/or FDG/ PET-CT fusion were performed to assess response to treatment after 2–3 courses of chemotherapy. Two patients received VAMP protocol (1 stage IA, 1 stage IIA), 1 received AV-PC (nonbulky stage IIA), 4 received COPP/ABV (3 stage IIA bulky, 1 stage IIIA nonbulky), 4 received ABVE-PC (2 stage IIB, 1 stage IIA bulky, 1 stage IIIA bulky), 2 received ABVD (1 stage IIA bulky, 1 stage IIIA), and 6 received escalated BEACOPP (1 stage IIIB, 3 stage IVA, 2 stage IVB). Results: Changes in HP levels were found to correlate with response to treatment for most of the children. At diagnosis, average HP level was 1019 pg/mL (range, 141–5733 pg/mL), decreasing at restaging to 588 pg/mL (range, 62–3267 pg/mL) (p =. 034). At diagnosis, the average HP of the 16 patients in CR or VGPR was 1104 pg/mL; it had decreased at restaging to 586 pg/mL (p =. 032). At diagnosis, the average HP level for the 3 patients with TP or PR was 1704 pg/mL; it had increased to 1938 pg/mL at restaging (p =. 166). Due to the small number of patients, no correlation was observed between HP levels at diagnosis, staging, or any other clinical prognostic factor. Conclusions: Changes in plasma HP levels correlated with response to treatment for children diagnosed with Hodgkin lymphoma. This provides a rationale for exploring clinical interest in plasma heparanase measurements of a larger group, using the test for clinical trials of antiangiogenic therapies.

Heparanase 2 Attenuates Head and Neck Tumor Vascularity and Growth

The endoglycosidase heparanase specifically cleaves the heparan sulfate (HS) side chains on proteoglycans, an activity that has been implicated strongly in tumor metastasis and angiogenesis. Heparanase-2 (Hpa2) is a close homolog of heparanase that lacks intrinsic HS-degrading activity but retains the capacity to bind HS with high affinity. In head and neck cancer patients, Hpa2 expression was markedly elevated, correlating with prolonged time to disease recurrence and inversely correlating with tumor cell dissemination to regional lymph nodes, suggesting that Hpa2 functions as a tumor suppressor. The molecular mechanism associated with favorable prognosis following Hpa2 induction is unclear. Here we provide evidence that Hpa2 overexpression in head and neck cancer cells markedly reduces tumor growth. Restrained tumor growth was associated with a prominent decrease in tumor vascularity (blood and lymph vessels), likely due to reduced Id1 expression, a transcription factor highly implicated in VEGF-A and VEGF-C gene regulation. We also noted that tumors produced by Hpa2-overexpressing cells are abundantly decorated with stromal cells and collagen deposition, correlating with a marked increase in lysyl oxidase expression. Notably, heparanase enzymatic activity was unimpaired in cells overexpressing Hpa2, suggesting that reduced tumor growth is not caused by heparanase regulation. Moreover, growth of tumor xenografts by Hpa2-overexpressing cells was unaffected by administration of a mAb that targets the heparin-binding domain of Hpa2, implying that Hpa2 function does not rely on heparanase or heparan sulfate. Cancer Res; 76(9); 2791-801. ©2016 AACR.