Elucidating mechanisms that regulate the expression of the heparanase homolog, Hpa2

Introduction. Heparanase activity is strongly implicated in tumor angiogenesis and metastasis attributed to remodeling of the subepithelial and subendothelial basement membranes. Augmented level of heparanase was documented in an increasing number of human carcinomas and hematological malignancies. In many cases, heparanase induction correlated with increased tumor metastasis, vascular density, and shorter survival rate post-operation, thus providing strong clinical support for the pro-tumorigenic function of the enzyme, making it an attractive target for the development of anti-cancer drugs. Heparanase homolog termed heparanase 2 (Hpa2) was cloned based on sequence similarity. Unlike heparanase, Hpa2 lacks intrinsic heparan sulfate (HS)-degrading activity, the hallmark of heparanase, but retains the capacity to bind heparin/HS. In fact, Hpa2 exhibits even higher affinity towards heparin/HS than heparanase, suggesting that Hpa2 may inhibit heparanase activity by competition for the HS substrate. Clinically, Hpa2 expression was markedly elevated in head and neck carcinoma patients, correlating with prolonged time to disease recurrence and inversely correlating with tumor cell dissemination to regional lymph nodes, suggesting that Hpa2 functions as a tumor suppressor. Similarly, Hpa2 was not detected in normal ducts of the pancreas but appeared to be expressed at high levels by some pancreatic ductal carcinomas. We hypothesized that Hpa2 induction in various tumors is due to stress conditions that tumor cells often experience in a fast-growing tumor lesion.

Methods. Pancreatic (Panc-O1, ASPC), head and neck (FaDu) and ovarian (Hey, SKOV-3) cells were exposed to ER-stress (by Thapsigargin), hypoxia or both and Hpa2 expression was evaluated by qPCR. We also examined Hpa2 expression in a panel of gastric and ovarian carcinoma cell lines in correlation with p53 status (wild-type vs mutant). We constructed a reporter gene composed of Hpa2 promoter fused with luciferase coding sequence that will enable the identification of regions and sequences that mediate Hpa2 gene regulation.

Results. ER stress and hypoxia each elicits modest, 3-5 fold increase in Hpa2 expression. However, combining ER stress and hypoxia resulted in a synergistic effect with an over 100-fold increase in Hpa2 expression. Moreover, we found that cells in which p53 is mutated are endowed with a much lower Hpa2 expression than cells carrying wild-type p53. Indeed, treatment with Nutlin that dissociates p53 from MDM2 and increases its activity resulted in increased Hpa2 expression.

Conclusions. We have identified, for the first time, mechanisms that regulate Hpa2 expression and will thereby assist in deciphering the function of Hpa2 in tumor growth.