Angiogenesis plays a critical role in breast cancer and is an integral part of tumorigenesis and metastasis. Growing tumors produce both angiogenic and angiostatic factors that regulate endothelial cell function. Many angiostatic factors, e.g., angiostatin and endostatin, are heparin-binding, cleaved products of large proteins which exhibit anti-tumorigenic activity by inhibiting neovascularization. Prolactin (PRL) is a 23 kDa pituitary hormone whose main target is the breast. Its N-terminal fragment, named 16K PRL, possesses angiostatic activity both in vitro and in vivo and its function is conserved across species. However, the ability of 16K PRL to arrest tumor growth and metastases has not been demonstrated. We have confirmed that proteolytically-cleaved rat PRL (rPRL) suppresses basal and FGF-stimulated proliferation of human and bovine endothelial cells. We showed that the metastatic MDA-MB-435 human breast cancer cells, transiently transfected with mammalian expression vectors, produce and secrete 16K and 23K human PRL (hPRL), and also demonstrated that baculoviral-infected insect cells secreted 16K PRL with angiostatic activity. In vivo tumor models for testing the angiostatic action of 16K PRL, using athymic mice, have also been established. Our hypothesis is: 16K PRL is a potent angiostatic factor which acts on endothelial cells via distinct receptors. Treatment of breast cancer with 16K PRL should suppress growth of primary tumors and metastases by reducing their blood supply. The first specific aim will determine whether 16K hPRL arrests growth of grafted MDA-MB-435 cells stably expressing 16K hPRL compared with those expressing 23K hPRL or containing empty vector. The second specific aim will examine whether 16K hPRL is generated by human tumor xenografts and determine the effects of tumor-produced 16K hPRL on blood vessel density, cell proliferation and apoptosis. The third specific aim will generate recombinant 16K hPRL in insect cells and examine its biochemical properties, angiostatic activity and binding to tumors and endothelial cells. The fourth specific aim will determine whether 16K hPRL suppresses growth of metastases in grafted athymic mice, using three alternative delivery methods: daily injections of recombinant 16K PRL, gene transfer via intramuscular injections of 16K hPRL plasmid, or implantation of 16K PRL-expressing COS cells encapsulated in hollow fibers.
