Metastasis is the principal cause of death in breast cancer patients. Hence, short of prevention of this cancer, no achievement would confer greater benefit on patients than methods to prevent metastasis. Our laboratory has recently published a novel mechanism by which blood-borne breast cancer cells arrest in the lung vasculature, extravasate, and colonize their most preferred organ, the lungs. Adhesion is between endothelial dipeptidyl peptidase IV (DPP IV) and fibrillar fibronectin assembled on breast cancer cell surfaces during hematogenous spread. The present proposal is designed to further characterize this binding mechanism, to explore its role in mediating extravasation, and colonization of the lungs and, ultimately, to develop novel anti-adhesion strategies in the treatment of breast cancer metastasis. Our first task will be to clone and sequence endothelial DPP IV from a cDNA library prepared from rat lung capillary endothelium. Using cDNAs from endothelial DPP IV and rat plasma fibronectin, we shall next determine the binding domains of the receptor/ligand pair. The strategy will be to generate a series of fusion proteins that cover the length of the interacting proteins followed by PCR-targeted mutagenesis once a fusion protein harboring the binding domain is identified. Peptides representing the binding domain will be tested for their ability to inhibit tumor cell binding to DPP IV and lung metastasis. The next two aims will test mechanisms of assembly and regulation of pericellular, fibrillar fibronectin by breast cancer cells. First, a series of recombinant fibronectins that have critical deletions in their matrix assembly domain will be generated in the baculovirus/insect cell expression system and their effect on DPP IV binding and transendothelial migration assayed. Next, the regulation of the integrin alpha5beta1 in immobilizing a fibronectin scaffold on cancer cells to allow self- association into fibrils will be tested in non-metastatic breast cancer cells that do not express this integrin. Regulatory cues exerted on this integrin by environmental will be analyzed. Finally, the role of DPP IV in initiating a signaling cascade in tumor cell-stimulated endothelial cells that leads to endothelial retraction, basement membrane degradation, and extravasation will be examined. These data will contribute to a better understanding of lung metastasis of breast cancer and provide the backbone in developing new therapies in cancer metastasis.
Showing the most recent 10 out of 11 publications
