Hua-Quan Miao

Heparanase Enhances Syndecan-1 Shedding A NOVEL MECHANISM FOR STIMULATION OF TUMOR GROWTH AND METASTASIS

When shed from the cell surface, the heparan sulfate proteoglycan syndecan-1 can facilitate the growth, angiogenesis, and metastasis of tumors. Here we report that tumor cell expression of heparanase, an enzyme known to be a potent promoter of tumor progression and metastasis, regulates both the level and location of syndecan-1 within the tumor microenvironment by enhancing its synthesis and subsequent shedding from the tumor cell surface. Heparanase regulation of syndecan-1 is detected in both human myeloma and breast cancer cell lines. This regulation requires the presence of active enzyme, because mutated forms of heparanase lacking heparan sulfate-degrading activity failed to influence syndecan-1 expression or shedding. Removal of heparan sulfate from the cell surface using bacterial heparitinase dramatically accelerated syndecan-1 shedding, suggesting that the effects of heparanase on syndecan-1 expression by tumor cells may be due, at least in part, to enzymatic removal or reduction in the size of heparan sulfate chains. Animals bearing tumors formed from cells expressing high levels of heparanase or animals transgenic for heparanase expression exhibited elevated levels of serum syndecan-1 as compared with controls, indicating that heparanase regulation of syndecan-1 expression and shedding can occur in vivo and impact cancer progression and perhaps other pathological states. These results reveal a new mechanism by which heparanase promotes an aggressive tumor phenotype and suggests that heparanase and syndecan-1 act synergistically to fine tune the tumor microenvironment and ensure robust tumor growth.

Inhibition of heparanase activity and tumor metastasis by laminarin sulfate and synthetic phosphorothioate oligodeoxynucleotides.

Heparanase activity correlates with the metastatic potential of tumor cells. Moreover, the anti‐metastatic effect of non‐anti‐coagulant species of heparin and certain sulfated polysaccharides was attributed to their heparanase‐inhibiting activity. We investigated the effect of a chemically sulfated polysaccharide (laminarin), consisting primarily of beta‐1,3 glucan (sodium laminarin), and of synthetic phosphorothioate oligodeoxynucleotides, primarily phosphorothioate homopolymer of cytidine (SdC28), on heparanase activity and tumor metastasis. Investigation of the ability of tumor cells to degrade heparan sulfate in intact extracellular matrix revealed that heparanase activity expressed by B16‐BL6 mouse melanoma cells and 13762 MAT rat mammary adenocarcinoma cells was effectively inhibited by LS (50% inhibition at 0.2–1 μg/ml), but there was no inhibition by sodium laminarin up to a concentration of 50 μg/ml. Complete inhibition of the melanoma heparanase was obtained in the presence of 0.1 μM SdC28. A single i.p. injection of laminarin sulfate, but not of sodium laminarin, before i.v. inoculation of the melanoma or breast‐carcinoma cells inhibited the extent of lung colonization by the tumor cells by 80 to 90%. Similar inhibition was exerted by 0.1 μM SdC28. At the effective concentrations, both compounds had a small effect on proliferation of the tumor cells and on growth of the primary tumors in vivo. These results further emphasize the involvement of heparanase in tumor metastasis and the potential clinical application of diverse heparanase‐inhibiting molecules such as sulfated polysaccharides and synthetic polyanionic molecules. Int. J. Cancer 83:424–431, 1999.

Development of heparanase inhibitors for anti-cancer therapy.

Heparanase is an endo-beta-D-glucuronidase that degrades heparan sulfate glycosaminoglycan side chains of the proteoglycans in extracellular matrix and basement membrane. Heparanase enzymatic activity is important in the promotion of tumor angiogenesis, primary tumor growth, invasion, and metastasis. Expression of heparanase in many tumor types conversely correlates with prognosis. Much progress has been made in studying the regulation of heparanase expression, processing and activation. The interaction between heparanase and its substrate heparan sulfate has been well characterized. The fact that heparanase was identified as the single predominant heparan sulfate-degrading enzyme in human cancer sparked considerable interest in developing heparanase inhibitors for potential therapeutic applications. Recent progress in drug development led to several classes of heparanase inhibitors, including chemically modified natural products, small molecule inhibitors, and antibodies. Some of these inhibitors have demonstrated potent activities to inhibit tumor angiogenesis, tumor progress, or tumor metastasis. A leading compound, PI-88, is currently being evaluated in clinical phase II trials in patients with melanoma, liver, or lung cancers. This review summarizes the recent progress in heparanase biochemical research and the development of heparanase antagonists as novel anti-cancer therapeutics.

Halofuginone: a potent inhibitor of critical steps in angiogenesis progression

We have previously demonstrated that halofuginone, a low molecular weight quinazolinone alkaloid, is a potent inhibitor of collagen α1(I) and matrix metalloproteinase 2 (MMP‐2) gene expres¬sion. Halofuginone also effectively suppresses tu¬mor progression and metastasis in mice. These re¬sults together with the well‐documented role of extracellular matrix (ECM) components and matrix degrading enzymes in formation of new blood ves¬sels led us to investigate the effect of halofuginone on the angiogenic process. In a variety of experimen¬tal system, representing sequential events in the angiogenic cascade, halofuginone treatment resulted in profound inhibitory effect. Among these are the abrogation of endothelial cell MMP‐2 expression and basement membrane invasion, capillary tube formation, and vascular sprouting, as well as depo¬sition of subendothelial ECM. The most conclusive anti‐angiogenic activity of halofuginone was demon‐strated in vivo (mouse corneal micropocket assay) by showing a marked inhibition of basic fibroblast growth factor (bFGF) ‐induced neovascularization in response to systemic administration of halofuginone, either i.p. or in the diet. The ability of halofuginone to interfere with key events in neovascularization, together with its oral bioavailability and safe use as an anti‐parasitic agent, make it a promis¬ing drug for further evaluation in the treatment of a wide range of diseases associated with pathological angiogenesis.—Elkin, M., Miao, H.‐Q., Nagler, A., Aingorn, E., Reich, R., Hemo, I., Dou, H.‐L., Pines, M., Vlodavsky, I. Halofuginone: a potent inhibitor of critical steps in angiogenesis progression. FASEB J. 14, 2477–2485 (2000)

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.

Involvement of the VEGF receptor 3 in tubular morphogenesis demonstrated with a human anti-human VEGFR-3 monoclonal antibody that antagonizes receptor activation by VEGF-C

In this report we utilize a novel antagonist antibody to the human VEGFR-3 to elucidate the role of this receptor in in vitro tubular morphogenesis of bovine and human endothelial cells (EC cells) induced by VEGF-C. The antibody hF4-3C5 was obtained by panning a human phage display library on soluble human VEGFR-3. The binding affinity constant of hF4-3C5 significantly exceeds that of the interaction of VEGFR-3 with VEGF-C. hF4-3C5 strongly inhibits the binding of soluble VEGFR-3 to immobilized VEGF-C and abolishes the VEGF-C-mediated mitogenic response of cells that expresses a chimeric human VEGFR-3-cFMS receptor. In fluorescence experiments, hF4-3C5 reactivity is observed with human lymphatic endothelial cells (LECs) and human umbilical vein endothelial cells (HUVECs). Binding of hF4-3C5 shows that about half of bovine aortic endothelial (BAE) cells express VEGFR-3 and cells in this subpopulation are primarily responsible for the chemotactic response to the mature form of VEGF-C (VEGF-CΔNΔC). This response was strongly inhibited by the addition of hF4-3C5. In vitro tube formation by BAE cells induced by VEGF-CΔNΔC was reduced by greater than 60% by hF4-3C5 whereas the response to VEGF165 was unaffected. Addition of hF4-3C5 together with an antagonist antibody to VEGFR-2 completely abolished the response to VEGF-CΔNΔC. Similar results were obtained with HUVECs. Together, these findings point to a role for VEGFR-3 in vascular tubular morphogenesis and highlight the utility of hF4-3C5 as a tool for the investigation of the biology of VEGFR-3.

A recombinant, fully human, bispecific antibody neutralizes the biological activities mediated by both vascular endothelial growth factor receptors 2 and 3

Vascular endothelial growth factors (VEGF) and their receptors (VEGFR) have been implicated to play important roles in tumor-associated angiogenesis and lymphangiogenesis, and hence in tumor growth and metastasis. We previously produced a number of fully human antibodies directed against VEGF receptor 2 (VEGFR2) and VEGF receptor 3 (VEGFR3) and showed that these antibodies are capable of inhibiting growth factor (VEGF and VEGF-C)-induced receptor activation, migration, and proliferation of human endothelial cells. In this report, we constructed and produced a bispecific antibody, a diabody, using the variable domain genes of two neutralizing antibodies, IMC-1121 to VEGFR2 and hF4-3C5 to VEGFR3. The diabody binds to both VEGFR2 and VEGFR3 in a dose-dependent manner, and blocks interaction between VEGF/VEGFR2, VEGF-C/VEGFR2, and VEGF-C/VEGFR3. In cell-based assays, the diabody neutralized both VEGF and VEGF-C-stimulated activation of VEGFR2, VEGFR3, and p44/p42 mitogen-activated protein kinase in endothelial cells. Furthermore, the diabody was able to inhibit both VEGF and VEGF-C-induced migration of endothelial cells. Taken together, our results suggest that a dual blockade of both VEGFR2 and VEGFR3 simultaneously may represent a more potent approach to effective cancer therapy.

A synthetic glycosaminoglycan mimetic (RGTA) modifies natural glycosaminoglycan species during myogenesis.

Crucial events in myogenesis rely on the highly regulated spatiotemporal distribution of cell surface heparan sulfate proteoglycans to which are associated growth factors, thus creating a specific microenvironment around muscle cells. Most growth factors involved in control of myoblast growth and differentiation are stored in the extracellular matrix through interaction with specific sequences of glycosaminoglycan oligosaccharides, mainly heparan sulfate (HS). Different HS subspecies revealed by specific antibodies, have been shown to provide spatiotemporal regulation during muscle development. We have previously shown that glycosaminoglycan (GAG) mimetics called RGTA (ReGeneraTing Agent), stimulate muscle precursor cell growth and differentiation. These data suggest an important role of GAGs during myogenesis; however, little is yet known about the different species of GAGs synthesized during myogenesis and their metabolic regulation. We therefore quantified GAGs during myogenesis of C2.7 cells and show that the composition of GAG species was modified during myogenic differentiation. In particular, HS levels were increased during this process. In addition, the GAG mimetic RGTA, which stimulated both growth and differentiation of C2.7 cells, increased the total amount of GAG produced by these cells without significantly altering their rate of sulfation. RGTA treatment further enhanced HS levels and changed its sub-species composition. Although mRNA levels of the enzymes involved in HS biosynthesis were almost unchanged during myogenic differentiation, heparanase mRNA levels decreased. RGTA did not markedly alter these levels. Here we show that the effects of RGTA on myoblast growth and differentiation are in part mediated through an alteration of GAG species and provide an important insight into the role of these molecules in normal or pathologic myogenic processes.