The cyclooxygenase (Cox) enzyme catalyzes the formation of prostanoids. Two genes, named Cox-l and -2 encode this enzyme; Cox-l is ubiquitously and constitutively expressed, whereas Cox-2 is induced as an immediate-early gene in response to growth factors, tumor promoters and carcinogens. The Cox-2 enzyme is overexpressed in various tumors (including mammary cancers), and deletion of the Cox-2 gene reduced the incidence and size of polyps in the Miii mouse model of intestinal neoplasia. These data suggest that inhibition of Cox-2 by non-steroidal anti-inflammatory drugs is a major mechanism for the cancer preventive activity of these drugs. This proposal is based on the hypothesis that the aberrantly expressed Cox-2 enzyme is a critical and sufficient inducer of tumorigenesis in the mammary gland. Further, we will test the hypothesis that prostanoid-dependent and -independent mechanisms contribute to mammary tumorigenesis by inhibiting cell death as well as promoting angiogenesis.
In Specific Aim I, we will derive transgenic mice overexpressing human Cox-2 in the mammary glands using the murine mammary tumor virus (MMTV) promoter. The role of dietary n-6 fatty acids, cooperativity with other mammary oncogenes (c-myc, Ha-Ras and cyclin Dl) will be assessed by deriving bigenic mice and quantifying tumorigenesis. Prostanoid synthesis by mammary tissues and tumors will be quantified. Mammary gland precocious differentiation, apoptosis during involution and tumorigenesis will be assessed.
In Specific Aim II, we will critically test the hypothesis that prostanoid-dependent and/or independent mechanisms are involved in Cox-2-induced tumorigenesis. Cox inhibitors will be administered to determine the requirement for prostanoid synthesis. We will attempt to test if PGE2 signaling via the EP family of G-protein coupled receptors is involved, In addition, we will test if PGI/2 PPAR signaling is involved. The role of peroxidase activity of the Cox-2 enzyme will be assessed by deriving trangenic mice with a mutant (S516Q) Cox-2 enzyme, which only possesses only the peroxidase activity.
In Specific Aim III, we will further characterize the effect of Cox-2 overexpression on apoptosis of the mammary epithelial cells and angiogenesis of the stroma. Mediators of apoptosis will be characterized with respect to activation by the prostanoids and Cox enzyme activity. Similarly, secreted angiogenic factors and their receptors will be characterized. We will define signal transduction mechanisms involved in these processes. Finally, we will employ a mass-spectrometry based state-of-the-art proteomic technology to identify novel polypeptides regulated by Cox-2 overexpression in the mammary gland, with a focus on mediators of apoptosis and angiogenesis.
