Spatial and temporal heparanase expression in colon mucosa throughout the adenoma-carcinoma sequence

Abstract

Heparanase is a mammalian endo-b-D-glucuronidase that cleaves heparan sulfate side chains at a limited number of sites. Such enzymatic activity is thought to participate in degradation and remodeling of the extracellular matrix and to facilitate cell invasion associated with tumor metastasis, angiogenesis and inflammation. Traditionally, heparanase activity was well correlated with the metastatic potential of a large number of tumor-derived cell types. More recently, heparanase upregulation has been documented in an increasing number of primary human tumors, correlating with poor postoperative survival and increased tumor vascularity. Here, we employed antiheparanase 733 polyclonal antibody that preferentially recognizes the 50 kDa active heparanase subunit over the 65 kDa proenzyme, as well as anti-heparanase 92.4 monoclonal antibody that recognizes both the latent and the active enzyme, to follow heparanase expression, processing and localization throughout the adenoma-carcinoma transition of the colon epithelium. Normal (nondysplastic) mucosa of the large bowel near epithelial neoplasms, as well as areas of mild dysplasia in adenomas, exhibited a strong reactivity with antibody 733 that became even stronger in foci of moderate dysplasia. Interestingly, although reactivity with antibody 733 was markedly reduced in severe dysplasia and in colorectal carcinoma, response to antibody 92.4 exhibited the opposite trend and staining intensities increased in parallel with tumor stage, the highest being in carcinoma cells. Involvement of latent heparanase (detected by 92.4, but not by 733 antibody) in tumor progression was suggested by activation of the Akt/PKB signal transduction pathway upon heparanase overexpression or exogenous addition to HT29 human colon carcinoma cells. These results suggest that heparanase expression is induced during colon carcinogenesis, and that its processing, conformation and localization are tightly regulated during the course of colon adenoma-carcinoma progression. Heparanase is an endoglycosidase that specifically cleaves heparan sulfate side chains of heparan sulfate proteoglycans. 1,2 Heparan sulfate proteoglycans consist of a protein core to which several heparan sulfate side chains are covalently attached. These complex macromolecules are highly abundant in the extracellular matrix and are thought to play an important structural role, contributing to extracellular matrix integrity and insolubility. 3 In addition, heparan sulfate side chains can bind to a variety of biological mediators such as growth factors, cytokines and chemokines, 4,5 thus functioning as a readily available reservoir that can be liberated upon local or systemic cues. Moreover, heparan sulfate proteoglycans on the cell surface participates directly in signal-transduction cascades by potentiating the interaction between certain growth factors and their receptors. 6-9 Heparan sulfate-degrading activity is thus expected to affect several fundamental aspects of cell behavior under normal and clinical settings. 1,2 Tr...

Heparanase expression by Barrett's epithelium and during esophageal carcinoma progression

“…In Barrett's and low-grade dysplasia, heparanase appeared punctuated and was localized to the basal aspect of the epithelium (Figure 1b, c). Interestingly, a similar staining pattern was noted in adenoma of the colon and in inflammatory bowel disease specimens, 14,26 both representing premalignant conditions. In contrast, heparanase appeared more diffusely distributed in the cell cytoplasm of highgrade dysplasia (Figure 1d) and adenocarcinoma specimens (Figure 1e) representing, possibly, heparanase protein that is more readily secreted.…”

“…In these studies, heparanase was not detected in normallooking epithelia, whereas high levels of expression were observed in colon and gastric carcinomas. [13][14][15][16][17] Heparanase induction has similarly been observed in carcinoma of the esophagus, and its expression correlated with tumor malignancy and poor survival. [18][19][20][21] Although heparanase upregulation and its pro-malignant features are well documented, the timing of its induction in the course of tumor development is less investigated and understood.…”

Heparanase upregulation by colonic epithelium in inflammatory bowel disease

“…14 Heparanase expression by the gastrointestinal tract has been documented by employing immunostaining, RT-PCR and heparanase activity analyses. [15][16][17] In these studies, heparanase was not detected in normal looking mucosa, whereas high levels of expression were detected in primary and metastatic colon carcinoma. [15][16][17] The involvement of heparanase in inflammatory processes typical for the gastrointestinal tract has not been so far examined.…”

“…No staining of heparanase was detected in normal colon tissue (Table 2, Figure 2a and b), in agreement with previous findings. 15,17 Similarly, no detectable heparanase staining was evident in infectious colitis specimens (Table 2, Figure 2d and e). In contrast, heparanase staining was markedly increased in Crohn's disease (Table 2, Figure 2g and h) and ulcerative colitis (Table 2, Figure 2j and k) specimens.…”

“…17,20 Briefly, slides were deparaffinized, rehydrated and endogenous peroxidase activity was quenched (30 min) by 3% hydrogen peroxide in methanol. Slides were then subjected to antigen retrieval by boiling (20 min) in 10 mM citrate buffer, pH 6.…”