Human Heparanase: A Molecular Determinant of Tumor Metastasis and Angiogenesis
Human Heparanase: A Molecular Determinant of Tumor Metastasis and Angiogenesis
Both benign and malignant tumors grow in an uncontrolled way, but only malignant tumors cells are capable of invading surrounding tissues and traveling to distant organs (i.e., metastasizing). It is this ability that makes cancer so tenacious and deadly. Metastatic cancer cells must be able to breach barriers, such as the extracellular matrix (ECM) or the basement membranes (BM). An understanding of the molecular basis for this invasion capability could result in therapies that block the transition of a tumor from the benign to the malignant phenotype.
Two of the essential processes required for metastasis are angiogenesis, which leads to blood vessel formation that feeds tumors, and ECM and BM degradation, which result in local invasion by tumor cells. ECM and BM are rigid structures formed from macromolecules such as type IV collagen, laminin, entactin, nidogen, fibronectin, and proteoglycans, one class of which is heparan sulfate proteoglycans (HSPG).1 HSPG metabolism, as related to tumor metastasis, is the focus of this article.
Isolating the Heparanase Gene
We know that HSPGs play a central role in embryonic morphogenesis, angiogenesis, neurite outgrowth, and tissue repair.2 ECM and BM HSPGs also provide a readily available storage depot for growth factors and cytokines. Since HSPGs now are recognized as active biological modulators, their degradation at the level of heparan sulfate (HS) chains is expected to have significant regulatory consequences in cancer metastasis. In previous work, researchers cloned several genes for proteases, the enzymes that cancer cells produce to degrade the protein portion of ECM and BM. However, substantial evidence has accumulated over the past 20 years indicating that metastatic tumors cells, but not their nonmetastatic counterparts, significantly overexpress heparanase enzymatic activity.3 Heparanase is responsible for HS degradation at specific intra-chain sites, resulting in the formation of HS fragments of discrete molecular weight, which identify the enzyme as an endo-b-D-glucuronidase.4,5 Additional findings of increased heparanase activity in tissue specimens from cancer patients with metastatic disease have confirmed its relevance in tumor metastasis.3
Although these phenomena are well documented, it has taken 20 years to isolate the heparanase gene because of the enzyme’s instability and the difficulty in designing specific, rapid, and quantitative assays. Only recently, several groups have reported the successful isolation and cloning of human heparanase as the first example of cloning a mammalian HS-degradative enzyme.6-9 Furthermore, the isolated cDNA sequences derived from normal or metastatic (human placenta, platelets) cells represent the same gene. Although metastasizing cancer cells can produce as many as 15 different matrix-digesting proteases, this finding is of unique importance because it indicates that there is only one heparanase. Therefore, if heparanase inhibition occurs (and recent evidence indicates that it can be inhibited effectively), other heparanases should not be around to cover for it.10 What’s more, in addition to inhibiting the cancer cells’ ability to roam, blocking heparanase also hinders angiogenesis, as the enzyme aids in the penetration of new tissue at the leading edge of the neovascularization. Heparanase can achieve this either directly, by affecting endothelial cell migration, or indirectly, by mobilizing potent angiogenic factors (i.e., basic fibroblast growth factor [bFGF] or vascular endothelial growth factor [VEGF]) that are stored in their inactive forms within the HS chains.
Now that the human heparanase cDNA sequence is available, with monoclonal antibodies and inhibitors having been developed recently, more direct strategies can be formulated to demonstrate heparanase’s involvement in metastasis at each level: gene, antigen, and pure enzyme.
Heparanase and Melanoma Progression
We have focused on heparanase’s role in invasion and angiogenesis of malignant melanoma, pertaining to its progression to the brain-metastatic phenotype. Our hypothesis is that brain metastases, so frequently associated with malignant melanoma, represent brain injury-related trauma in which a family of neurotrophic factors, neurotrophins (NT), and related receptors, notably p75NTR, play relevant roles in melanoma progression to the brain-metastatic phenotype.11-14 NT modulate brain invasion of melanoma cells and heparanase production.13-16 Mechanisms responsible for the progression of malignant melanoma to highly aggressive brain-metastatic disease remain largely unknown. Thus, heparanase can play important roles in melanoma progression, with the brain considered the ideal target organ because of the elevated NT production and the strong angiogenic properties of melanoma cells found in the brain.
Of equal importance, we have postulated roles for the normal cells present in the brain microenvironment, notably astrocytes, and their contributions in the brain-metastatic specificity of melanoma cells. To test these hypotheses, we employed purified astrocyte cultures as in vitro models.17 We found that astrocytes express both the heparanase transcript and the functional enzyme, the activity of which was upregulated by as much as four-fold by the prototypic NT, nerve growth factor (NGF).18 Co-incubation of astrocytes (or their conditioned medium) with brain-metastatic cells resulted in a superadditive effect on heparanase activity and up to an eight-fold increase of in vitro chemoinvasion using purified HSPG as substrate.18 These observations demonstrate that astrocytic interactions with melanoma cells can contribute significantly to the brain colonization of melanoma cells and alter invasion via heparanase and possibly other degradative enzyme-driven mechanisms. These observations also support the concept that mela-noma brain invasion results from establishing reciprocal circuits between the tumor cells and the normal glial cells present in the brain microenvironment.19 Following mechanical/chemical brain insults, increased NT/NTR presence is imperative in neuronal regeneration. These changes can be paralleled by brain invasive melanoma cells whose colonization within the brain microenvironment triggers NT production and heparanase secretion by surrounding astrocytes as a response to the invasion event. Melanoma cells overexpressing p75NTR can benefit from such a synergistic microenvironment in terms of survival, growth, and further invasion into the brain parenchyma.
Heparanase and Invasion Mechanisms
We also have provided direct evidence for heparanase functionality in invasion mechanisms. Transfection of the heparanase gene into nonmetastatic melanoma cells resulted in augmented (up to 14-fold) enzymatic activity that correlated well with increased in vitro invasion by transfected nonmetastatic cells.20 Heparanase mRNA also was found to be present in greater amounts (at least 10-fold) in melanoma specimens at various stages of tumor progression, with highest expression found in invasive melanoma tissues. Similar results have been obtained with a variety of tumors other than malignant melanoma such as neuroblastoma, cervix and colon carcinoma, and metastatic cancers of the breast and prostate.20
Conclusion
These data indicate that heparanase expression may be part of the cellular switch from non-invasive to invasive phenotypes. The elucidation of nucleotide and amino acid sequences of human heparanase represents an important achievement, removing barriers to better understanding heparanase’s roles in metastasis, as involved in the invasive and angiogenic steps.
Questions related to the potential benefit of the therapeutic suppression of heparanase in brain-metastatic melanoma, or metastasis in general, remain unanswered. Further studies combining newly developed heparanase probes with in vivo experimental metastasis and angiogenic assays will be useful in addressing these questions. Additionally, heparanase markers may allow the assessment of disease progression or response to therapy.
Finally, the availability of large quantities of recombinant enzyme and sensitive functional assays will facilitate the design, testing, and use of selective inhibitors in clinical trials. However, rigorous evaluation of possible adverse effects on normal physiological functions will be imperative because, as with many enzyme systems, balance is essential. For example, knowledge that bFGF signaling is facilitated when the factor is HS-bound
cautions that use of heparanase inhibitors may shift
the equilibrium from free bFGF to HS-bFGF, altering recycling and degradation pathways, and enhancing rather that inhibiting cellular activity. (Dr. Marchetti is an Assistant Professor in the Department of Neurosurgery, Health Science Center, The University of Texas-Houston.)
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