G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors with more than 1000 members yet only a few GPCRs have been found to be oncogenes. Among these, the protease-activated receptor 1 (PAR1) has been identified as a potent oncogene and confers invasive behavior to pre-cancerous breast cells. In this grant we test the hypothesis that PAR1 is critical for cancer-host communication by stimulating production of paracrine and angiogenesis factors in the tumor environment. We recently identified host fibroblast-derived matrix metalloprotease-1 (MMP-1) as a novel agonist that can cleave and activate PAR1 in breast and ovarian tumors. MMP-1 is highly expressed in stromal cells and is a predictive marker for poor prognosis in breast, colorectal and other tumors. Targeting PAR1 with the novel cell-penetrating pepducins described here blocks the pathway downstream of MMP-1 and receptor inhibiting cancer growth and invasion. PAR1 pepducins also resulted in pronounced reduction of stromal infiltration and angiogenesis of breast and ovarian cancers. We will test the hypothesis that stromal-derived PAR1 may have a distinct role from cancer cell-derived PAR1 in tumor biology and stimulation of paracrine factors that promote invasion and angiogenesis. We will utilize two cancer-stromal co-culturing model systems and in vivo cancer- stromal coimplantation xenografts. Activation of PAR1 (and PAR2) have been shown to produce IL-8, Gro-?, VEGF, IL-6 and GM-CSF in a variety of cell types including prostate cancer, but the role of PAR1 in paracrine communication in cancer has not been directly addressed. We hypothesize that activation of PAR1 on cancer cells leads to production of chemokines which stimulate endothelial cells resulting in increased angiogenesis and tumor growth. Pepducin technology will be used to define the role of endothelial chemokine CXCR1 and CXCR2 receptors in blood vessel formation and validate our preliminary data that PAR1 and potentially CXCR1/2 pepducins can block angiogenesis and extend survival in ovarian and breast xenograft animal models. Lastly, we will follow-up our recent discovery of a novel PAR1-effector, BicD1, which acts as a suppressor of PAR1-dependent migration and invasion of breast cancer cells. Knock-down of BicD1 expression greatly prolongs mitogen-activated kinase signaling suggesting that BicD1 may regulate MAPK phosphatase (MKP) activity in breast cancer cells. We will test the hypothesis that upregulation of PAR1 expression or stimulation of PAR1 by MMP-1 or thrombin regulates BicD1 and MKP expression and that these downstream effectors in turn control MAPK-dependent invasion and proliferation of breast cancer. The pepducin approach has the prospect to significantly change our understanding of the role of cross-talk between receptors such PAR1 and CXCR1/2 in cancer growth and blood vessel formation. As envisioned, these studies will develop the first inhibitors of these invasion and chemokine receptors for the potential treatment of advanced breast and ovarian cancers. ? ? ? ? ? ?
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