RO1: A novel and targeted approach to inhibit invasion and angiogenesis

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DESCRIPTION (provided by applicant): Among many solid tumors, pancreatic cancer (PC) has the worst prognosis and it is the fourth leading cause of all cancer deaths in the United States, which could be partly due to the ability of PC cells to orchestrate in "turning-on the switch" for migration, invasion, angiogenesis and metastatic processes during the early course of the disease. The development of PC is strongly favored due to initiation of inflammatory events as suggested by the presence of extensive desmoplastic stroma (fibrogenesis) found in human PCs, partly contributed by PDGF over-expression, generation of reactive oxygen species (ROS) and activation of NF-:B. In addition, activation of EGFR and Notch-1 signaling also leads to the activation of NF-:B and, in turn, regulates NF-:B downstream signaling molecules such as COX-2, MMP-9, uPA and VEGF, among others, that are known to be causally associated with migration, invasion, angiogenesis and metastasis, ultimately leading to poor survival of patients diagnosed with PCs. While conducting experiments using a non-toxic dietary chemopreventive agent, 3,3'-diindolylmethane (DIM), especially BR-DIM (hereafter known as B-DIM, a formulated DIM with greater bioavailability supplied by BioResponse, Inc.), we found that B-DIM is a potent inhibitor of PDGF-D (a recently identified PDGF), EGFR, Notch-1 and NF-:B which, in turn, inhibits MMP-9, uPA, PGE2 and VEGF secretion in B-DIM treated cells. Based on our results, we hypothesize that the use of B-DIM will serve as a novel approach by which the processes of cell migration, invasion and angiogenesis could be inhibited. We will test our hypothesis by accomplishing the following three specific aims. In aim-1, we will thoroughly investigate the consequence of B-DIM treatment of PC cells in vitro by analyzing the effect of B-DIM on PDGF-D, EGFR, Notch-1, NF-:B, uPA, MMP-9, VEGF, COX-2 (PGE2) in preventing cell growth, tumor cell migration, invasion and apoptosis, and the effect of B-DIM on angiogenesis tested by using conditioned media from B-DIM treated cells in vitro and also by plug assay in vivo. In aim-2, we will investigate the molecular mechanism(s) and the specific contribution of PDGF-D, EGFR, Notch-1, NF-:B, uPA, MMP-9, VEGF and COX-2 in mediating the biological activity of B-DIM in PC cells in vitro by applying knock-down and over-expression approaches. In the third specific aim, we will test the in vivo anti-tumor activity of B-DIM using an orthotopic PC model in SCID mice as well as in transgenic mouse models, and test whether the anti- tumor activity could be correlated with the down-regulation of EGFR, NF-:B, uPA, MMP-9, VEGF, PDGF-D and COX-2 in tumor tissues. Moreover, we will test whether the inactivation of these signaling pathways could lead to sensitization of PC cells to conventional therapeutic agents. The completion of the proposed study will provide mechanism-based evidence in support of the use of B-DIM for preventing PC cell growth, migration, invasion and angiogenesis, and induction of apoptotic cell death. PUBLIC HEALTH RELEVANCE: Our results could aid in designing innovative preventive and/or therapeutic approaches for saving lives of patients diagnosed with this deadly disease. This project is focused on elucidating the mechanism by which a chemopreventive agent (B-DIM) could prevent pancreatic tumor invasion and angiogenesis, resulting in the inhibition of tumor progression. We hypothesize that B-DIM will down regulate PDGF-D signaling among others, which in turn will inactivate PDGF-D downstream signaling, resulting in the inhibition of cell growth, invasion and angiogenesis, and the inhibition of tumor progression. We will test our hypothesis by three specific aims using molecular approaches and animal model studies.