The overall objective of this proposal is to validate cyclic GMP phosphodiesterase (cGMP PDE) as a novel drug target for cancer chemoprevention. Previous studies have suggested that the chemopreventive efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) involve a cyclooxygenase (COX)-dependent and independent mechanism of action, although the precise target(s) and pathways responsible for their apoptosis inducing activity have not been well defined. In support of a COX-independent mechanism, structurally related drugs such as sulindac sulfone have been shown to have broad chemopreventive efficacy in clinical and preclinical studies without inhibiting COX-1 or -2 isozymes. Moreover, high dosages of NSAIDs and COX-2 inhibitors appear to be required for chemopreventive efficacy relative to their anti-inflammatory activity. The possibility of disassociating COX inhibitory activity from their antineoplastic activity has significant implications for developing safer and more efficacious drugs given that the chemopreventive efficacy of NSAIDs, including COX-2 selective inhibitors, is limited by COX-dependent toxicity. Recently, sulindac sulfone was reported to inhibit cGMP PDE and elevate intracellular cGMP levels to activate protein kinase G (PKG). This pathway has been linked to apoptosis induction by a novel mechanism involving PKG-mediated phosphorylation and degradation of beta-catenin to inhibit Tcf-dependent transcription of cell survival genes. We hypothesize that the chemopreventive activity of certain NSAIDs and COX-2 selective inhibitors is also mediated by cGMP PDE inhibition which may provide a highly selective mechanism for inducing apoptosis of neoplastic cells that harbor Apc or beta-catenin mutations. The proposed studies will focus on colon carcinogenesis that involves Apc gene mutations and can be inhibited by NSAIDs in a COX-independent manner. In vitro and in vivo studies are proposed to determine if cGMP PDE is responsible for the chemopreventive efficacy of NSAIDs and COX-2 inhibitors. Initial studies will determine if the growth inhibitory and apoptosis inducing activity of a panel of structurally diverse NSAIDs and COX-2 selective inhibitors correlate with cGMP PDE inhibition and will be compared to inhibitory effects on cAMP PDE as well as COX-1 and COX-2. The effects of PDE inhibitory NSAIDs (PiNs) on intracellular cGMP and cAMP levels will be measured to study potential isozyme selectivity in intact colon tumor cells. The activation of PKG and potential effects on PKA as well as a downstream pathway involving beta-catenin degradation and inhibition of Tcf-dependent transcription will be investigated. In vivo studies in the ApcMin mouse model of colon tumorigenesis will be performed to compare the efficacy of a PIN and a non-COX inhibitory cGMP PDE inhibitor. Finally, cGMP and cAMP levels will be measured in tumors and plasma samples from this study to determine if PDE inhibition occurs in vivo and the utility of cyclic nucleotides as biomarkers.
