Expression of Heparanase in Normal, Dysplastic, and Neoplastic Human Colonic Mucosa and Stroma

Abstract

The human heparanase gene, an endo-beta-glucuronidase that cleaves heparan sulfate at specific intrachain sites, has recently been cloned and shown to function in tumor progression and metastatic spread. Antisense digoxigenin-labeled heparanase RNA probe and monoclonal anti-human heparanase antibodies were used to examine the expression of the heparanase gene and protein in normal, dysplastic, and neoplastic human colonic mucosa. To our knowledge, this is the first systematic study of heparanase expression in human colon cancer. Both the heparanase 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-looking colon epithelium. Gradually increasing expression of heparanase was evident as the cells progressed from severe dysplasia through well-differentiated to poorly differentiated colon carcinoma. Deeply invading colon carcinoma cells showed the highest levels of the heparanase mRNA and protein associated with expression of both the gene and enzyme by adjacent desmoplastic stromal fibroblasts. A high expression was also found in colon carcinoma metastases to lung, liver, and lymph nodes, as well as in the accompanying stromal fibroblasts. Moreover, extracts derived from tumor tissue expressed much higher levels of the heparanase protein and activity as compared to the normal colon tissue. In all specimens, the heparanase gene and protein exhibited the same pattern of expression. These results suggest a role of heparanase in colon cancer progression and may have both prognostic and therapeutic applications.

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

“…[26][27][28]42 Heparanase upregulation has been noted in several primary human tumors, including colon carcinoma by employing in situ hybridization and immunostaining. 31 Moreover, heparanase upregulation in colon carcinoma correlated with increased lymph vessel and vascular infiltration and, most importantly, with reduced postoperative survival rate, 43 suggesting that heparanase expression levels may be considered as an important diagnostic parameter. The antibodies so far employed for heparanase detection do not distinguish, however, between the latent 65 kDa and active 50 kDa forms of the enzyme.…”

“…29 Anti-heparanase monoclonal antibody 92.4 has previously been characterized and shown to stain positively paraffin-embedded colon carcinoma specimens. 30,31 Antibody 1453 was raised in rabbits against the full-length 65 kDa latent heparanase protein. 26 Immunohistochemistry was performed essentially as described.…”

“…26 Immunohistochemistry was performed essentially as described. 31,32 Briefly, 5 mm sections were deparaffinized and rehydrated, followed by antigen retrieval (boiling in 10 mM citrate buffer, pH 6, for 10 min in a microwave oven). Sections were then incubated with 3% H 2 O 2 in absolute methanol for 30 min to inactivate endogenous peroxidase activity, washed with PBS and incubated with 10% goat serum to block nonspecific binding, followed by overnight incubation with the respective primary antibody (pAb 733 diluted 1:100, mAb 92.4 diluted 1:5 in blocking solution) at 41C.…”

“…By far, the strongest reactivity with antibody 92.4 was observed in the deep growing edge of the carcinoma (Figure 1e, arrowheads, Table 1; P50.05 compared to carcinoma at a more superficial level), in agreement with the proinvasive function of the heparanase enzyme. 2,15,30,31,35 In cell clusters, heparanase was noted to assume both diffuse cytoplasmic and membrane-like localization (Figure 1f, arrows), whereas invading single carcinoma cells exhibited mostly membrane-like localization of heparanase (Figure 1f, arrowheads).…”

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

“…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.…”