Proteoglycans in health and disease: new concepts for heparanase function in tumor progression and metastasis

Abstract

Heparanase is an endo-β-D-glucuronidase capable of cleaving heparan sulfate side chains at a limited number of sites, yielding heparan sulfate fragments of still appreciable size. Importantly, heparanase activity correlates with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of heparan sulfate cleavage and remodeling of the extracellular matrix and basement membrane underlying epithelial and endothelial cells. Similarly, heparanase activity is implicated in neovascularization, inflammation and autoimmunity, involving the migration of vascular endothelial cells and activated cells of the immune system. The cloning of a single human heparanase cDNA 10 years ago enabled researchers to critically approve the notion that heparan sulfate cleavage by heparanase is required for structural remodeling of the extracellular matrix, thereby facilitating cell invasion. Progress in the field has expanded the scope of heparanase function and its significance in tumor progression and other pathologies. Notably, although heparanase inhibitors attenuated tumor progression and metastasis in several experimental systems, other studies revealed that heparanase also functions in an enzymatic activity-independent manner. Thus, inactive heparanase was noted to facilitate adhesion and migration of primary endothelial cells and to promote phosphorylation of signaling molecules such as Akt and Src, facilitating gene transcription (i.e. vascular endothelial growth factor) and phosphorylation of selected Src substrates (i.e. endothelial growth factor receptor). The concept of enzymatic activity-independent function of heparanase gained substantial support by the recent identification of the heparanase C-terminus domain as the molecular determinant behind its signaling capacity. Identification and characterization of a human heparanase splice variant (T5) devoid of enzymatic activity and endowed with protumorigenic characteristics, elucidation of cross-talk between heparanase and other extracellular matrix-degrading enzymes, and identification of single nucleotide polymorphism associated with heparanase expression and increased risk of graft versus host disease add other layers of complexity to heparanase function in health and disease.

Figure 1

A. Immunohistochemical staining of heparanase in SCCHN tumor specimens Formalin-fixed, paraffin-embedded 5 micron sections of head & neck tumors were subjected to immunostaining of heparanase, applying anti-heparanase pAb #733. Shown are representative photomicrographs of positively stained specimens exhibiting cytoplasmic (Cyto, middle panel) and nuclear (Nuc, lower panel) heparanase localization. Normal looking tissue adjacent to the tumor lesion stained negative for heparanase (upper panel). Nuclear heparanase is associated with decreased levels of phospho-EGFR, lower lymph vessel density, and favorable prognosis of head & neck cancer patients (see text for details). B. Heparanase expression associates with tumor cell invasion into lymph vessels. Head & neck tumor specimen was stained with anti heparanase polyclonal (green, upper panel) and D2-40 monoclonal (a marker for human lymphatics; red, middle panel) antibodies, illustrating heparanase-positive tumor cells inside a lymphatic vessel lumen (merge, lower panel).

Figure 2. Heparanase splice variant, T5, endowed with pro-tumorigenic characteristics

A. Schematic structure of wild type (WT) and heparanase splice variant, T5. SP-signal peptide; glutamic acids residues 225 and 343 critical for heparanase enzymatic activity, are detonated (see text for details). B. Colony formation in soft agar. Control (Vo) heparanase (Hepa)-, and T5-infected myeloma (CAG, upper panels), pharynx (FaDu, second panels) and embryonic kidney (293, third panels) cells (5×10³ cells/dish) were mixed with soft agar and cultured for 3–5 weeks. CAG cells were similarly grown in the absence (DMSO; fourth panels) or presence of Src inhibitor (PP2, 0.4 nM; lower panels). Shown are representative photomicrographs of colonies at high (x100) magnification. C. Tumor xenograft development. Control (Vo), heparanase-, and T5-infected CAG myeloma cells were injected subcutaneously (1×10⁶/0.1ml). At the end of the experiment on day 37, tumors were harvested and photographed.

Figure 3. Immunohistochemical staining of esophageal specimens

A. Formalin-fixed, paraffin-embedded 5 micron sections of normal (upper panel), Barrett’s (second panel), low grade (third panel), high-grade (fourth panel), and adenocarcinoma (lower panel) esophageal biopsies were subjected to immunostaining of heparanase, applying anti-heparanase pAb #733 (left panels) or anti Ki-67, a marker of cell proliferation (right panels).