Survival, growth and spread of tumors depend upon an adequate blood supply ensured by neovascularization, cooption of existing vessels and stem cell differentiation into endothelial cells. Thus, inhibitors of angiogenesis may represent novel tools for the treatment of cancer. We observed that supernatants of EBV-immortalized cell generally contain inhibitors of endothelial cells proliferation. From supernatants of the lymphoblastic VDS-O cell line we purified an endothelial cell inhibitor, and identified it as a mixture of NH2-terminal fragments of calreticulin. The recombinant purified NH2-terminal fragments of calreticulin (amino acids 1-180) inhibited the proliferation of endothelial cells but not the proliferation of cells of other lineages, and suppressed angiogenesis in vivo. We have named this NH2-terminal domain of calreticulin vasostatin. When inoculated into athymic mice, vasostatin reduced growth of Burkitt lymphoma, colon carcinoma, breast adenocarcinoma, melanoma, rhabdomyosarcoma, lung adenocarcinoma and Wilms tumors. Full-length calreticulin, calreticulin lacking the NH2 terminal 1-120 amino acids, an internal calreticulin fragment encompassing amino acids 120-180, and an internal fragment encompassing amino acids 104-164 inhibited endothelial cell proliferation, angiogenesis, and tumor growth in vivo comparably to vasostatin. These results suggest that the antiangiogenic and antitumor activities of vasostatin reside in a domain that is accessible from the full-length calreticulin molecule and localized to an internal 61 amino acids fragment within the NH2 domain of calreticulin. Efforts aimed at clarifying the mechanims by which carleticulin and its active fragments inhibit endothelial cell proliferation have identified a laminin binding site within calreticulin 103-163. This binding of calreticulin to laminin specifically inhibits endothelial cell attachment to laminin-coated surfaces resulting in reduced endothelial cell survival and growth. In addition, we have identified specific calreticulin binding to endothelial cell membranes occurring independently from endothelial cell surface laminin expression. Thus, we have identified calreticulin and fragments of calreticulin N-domain as novel angiogenesis inhibitors that suppress tumor growth in preclinical tumor models.
