Colon cancer is the third leading killer of men and women in the U.S, and occurs much more frequently in patients with inflammatory bowel disease (IBD). Epidemiologic data suggests that non-steroidal anti- inflammatory (NSAID) drugs lower the risk of colon cancer, but the mechanism(s) responsible are not known. Mice lacking the G-protein alpha subunit Gia2 mount excessive, Th1-skewed inflammatory responses and spontaneously develop colitis, and then non-polypoid mucinous colon cancers. Since the causes of IBD in humans are unknown, the Giot2-/- model of IBD is useful for studying the regulation of mucosal inflammatory responses and colon cancer. In vitro studies by previous investigators identified a defect in release of arachidonic acid (AA) by cytoplasmic phospholipase A2 (cPLA2) in the absence of Gia2. AA is the precursor for multiple classes of eicosanoids including PGE2, which enhances oral tolerance and suppresses the induction of Th1 responses. We have shown that decreased AA release from colonic myofibroblasts (CMF) leads to less PGE2 synthesis, with corresponding decreases in colonic tissue levels of PGE2. Treatment with exogenous PGE2 analog attenuates colitis, arguing for the importance of arachidonate-derived mediators in regulating colonic inflammation. Gia2-/- mice develop colon cancer despite having lower mucosal levels of PGE2. cPLA2-derived AA also stimulates sphingomyelinase activity, enhancing ceramide production that induces apoptosis. We therefore hypothesize that decreased arachidonate release in colonic epithelium may therefore interfere with apoptosis, contributing to the development of colon cancer in Gia2-/- mice The long-term objective and major aims of this project are: A) to understand the mechanism by which a lack of Gioc2 inhibits cPLA2- mediated arachidonic acid release, and B) whether decreased arachidonate affects sphingolipid metabolism such that there is an inhibition of epithelial apoptosis that may predispose to the development of cancer.
In Aim 1 we use primary murine CMF to evaluate the signaling pathways that link Gia2 signaling to cPLA2 activation, translocation, and AA release.
In Aim 2 we examine how decreased arachidonate release affects different colonic sphingomyelinase activities, quantify changes in epithelial ceramide production, and determine how these changes affect the balance of colonic epithelial proliferation and apoptosis. Completion of these aims will provide mechanistic insight into how a lack of Gia2 signaling attenuates arachidonic acid release, with important consequences for mucosal immunity and colorectal neoplasia. Importantly, these animals also provide a useful in vivo model for addressing whether NSAID chemoprevention via COX inhibition is due to suppression of prostanoid production or elevation of mucosal free arachidonate levels. ? ? ?
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