Updated 3 September, 2001; 5 July, 1999

Metastatic cancer cells breach extracellular matrix (ECM) and basement membrane of the blood vessel to metastasize to the target organ (ectopic site). EMC consists of proteins embedded in carbohydrate complex (heparan sulfate peptidoglycans). As described in another article on Recent Aspects of Metastatic Cancer-Host Cross Talk and Targets for Drug Development, there are many proteases (about 15) isolated so far that break down the protein portions of these cell surface barriers, and some of them (metalloproteinases) have been extensively used as promising targets for developing drugs against metastasis. As to the carbohydrate portions of the surface barriers, heparan sulfate peptidoglycans in ECM or attached to cell surfaces interact not only with ECM constituents but also with a multitude of proteins (growth factors, enzymes, etc) via their heparan sulfate side chains. Consequently, heparan sulfate cleavage by an endoglycosidic enzyme "heparanase" could affect various biological processes. Also heparin that is an anticoagulant and a mixture of related acid mucosaccharides is known to inhibit metastasis, and these molecules may be further purified to separate anticoagulant from anti-metastasis activities (Vlodavsky I et al, 1994-95: uid=7657522). It was suggested that a heparanase that cleaves heparin may be important in the degradation of the extracellular matrix by invading cells, notably metastatic malignant cells and migrating leukocytes to extravasate through the vascular basal lamina. The heparanase as an endoglycosidase (molecular weight 45 kDa) was later purified from rat liver, human colonic carcinoma and human platelet (Freeman C et al, 1999: uid=10455023). The enzyme degraded both heparin and heparan sulfate to fragments of similar sizes. Together with additional similarities in peptide sequences and immunoreactivities, it appeared that quite a similar enzyme mediates heparanase activities in these mammalian tissues.

For further characterization of the heparanase activity, the substrate specificity was first defined for the heparanase from human hepatoma and platelet as to cleave the single beta-D-glucuronidic linkage of a heparin octasaccharide (an endo-beta-D-glucuronidase; Pikas DS et al, 1998: uid=9668050). Vlodavsky I et al (1999: uid=10395325) later successfully purified a 50-kDa heparanase from human hepatoma and placenta, and finally cloned cDNA and gene of the enzyme. The expression of the gene gave rise to 65-kDa and 50-kDa proteins, the latter of which is a N-terminally processed protein and 100-fold more active than the former. The heparanase mRNA and protein are preferentially expressed in metastatic cell lines, and human breast, colon and liver carcinomas. Hulett MD et al (1999: uid=10395326) also reported the cDNA cloning of a heparanase from the human platelet, which encodes a unique protein of 543 amino acids, and identified highly homologous sequences in activated mouse T cells and in a highly metastatic rat adenocarcinoma. Furthermore, the expression of heparanase mRNA in rat tumor cells correlated with their metastatic potential, namely, heparanase transfected into non-metastasizing cells (low-metastasizing murine T-lymphoma and melanoma cells) made them so as to massively colonize the lung and liver (Vlodavsky I et al, 1999: uid=10395325). Conversely, a heparanase inhibitor inhibited the metastasis, namely, in rats heparanase inhibitor PI-88 inhibited the metastasis of breast cancer cells to the lung by 90% (Parish CR et al, 1999: uid=10416607). Exhaustive studies of heparanase-like sequences in the database so far indicate that there may be only one heparanase sequence, consistent with the idea that this enzyme is the dominant endo-beta-D-glucuronidase in mammalian tissues involved in extracellular matrix degradation.

The expression of human heparanase gene and protein in normal colonic mucosa and colon cancer was systematically examined by the use of antisense heparanase RNA probe and monoclonal anti-human heparanase antibodies (Friedmann Y et al, 2000: uid=11021821). Both the gene and protein were expressed at early stages of neoplasia, already at the stage of adenoma, but were practically not detected in the adjacent normal colon epithelium. There was increasing expression of heparanase as the differentiation progressed to carcinoma, and deeply invading colon carcinoma cells showed highest levels of the heparanase mRNA. A high expression was also found in colon carcinoma metastasized to lung, liver, and lymph nodes, as well as in the accompanying stromal fibroblasts. The heparanase gene and protein exhibited the same pattern of expression in all specimens.

Heparanase inhibitor PI-88 briefly mentioned above is in fact a heparinomimetic phosphomannopentaose sulfate that is a structural minic of heparan sulfate and derived from pentasaccharide phosphate, 6-O-PO3H2-alpha-D-Man-(1 --> 3)-alpha-D-Man-(1 --> 3)-alpha-D-Man-(1 C 3)-alpha-D-Man-(1 --> 2)-D-Man. However, tetrasaccharide and disaccharide components are also present to some extent. This mannopentaose phosphate sulfate is a fraction from mild acid-catalysed hydrolysis of the extracellular phosphomannan of the yeast Pichia holstii (Ferro V et al, 2001: uid=11434376), and its large-scale production is now feasible. In addition to being a potent inhibitor of blood-borne metastasis (>90%), PI-88 inhibited the primary tumor growth of a highly invasive rat mammary adenocarcinoma by ca 50%, and metastasis to the draining lymph node by ca 40% (Parish CR et al, 1999: uid=10416607). Significantly, PI-88 also reduced the vascularity of tumors (angiogenesis) by ca 30%. It is likely that the heparan sulfate mimetic is a angiogenesis inhivitor as well as a heparanase inhibitor.

In addition to heparanase inhibitors from natural sources, it may now be feasible to design them by the use of modern concept and techniques in computational chemistry. Please go to Drug Design Approaches: Molecular Modeling, Computational Chemistry and Combinatorial Chemistry for conceptual and technical details. This notion comes from the recent finding of active site-residues of human heparanase (Hulett MD et al, 2000: uid=11123890). Similarities were identified between heparanase and several of the glycosyl hydrolases, including strong local identities to regions containing the critical active-site catalytic proton donor and nucleophile residues that are conserved in those hydrolases. On the basis of sequence alignments with a number of glycosyl hydrolases, Glu(225) and Glu(343) of human heparanase were identified as the likely proton donor and nucleophile residues, respectively. The substitution of these residues with alanine abolished the heparan sulfate-degrading capacity. In a race to develop heparanase inhibitors, it would now be of advantage to count on structure-based drug design and combinatorial chemistry, together with fast-advancing databases for quantitative structure activity/property relationship.

The findings described above provide direct evidence of an essential role of heparanase in metastasis, and would certainly help focus the future effort for anti-metastatic drugs on the enzyme, perhaps with possibly better chance of success than on the previous metalloproteinases. Of course, there remains a serious problem of how cancer cells already metastasized to the ectopic sites (target organs) may be blocked. It may be a totally different story to intervene tumor colonies growing inside the vascular vessel of the target organ than to block metastasis processes of malignant cancer. For the present we will wait and see how various heparanase inhibitors behave when employed in the clinical settings. At least initially the heparanase gene should serve as a specific probe for early detection of cancer metastasis. However, all in all, heparanase has emerged as a potential target for the development of anti-metastasis drugs.