This application continues our identification and dissection of pathways that are critical in the formation and modulation of intestinal cancer. Our approach utilizes genetic, nutritional and pharmacological modulation of intestinal tumorigenesis in the mouse initiated either by an Apc mutation, or, in a new model made by us, by targeted inactivation of Muc2, the gene that encodes the major gastrointestinal mucin.
Aim 1 completes analysis of modulation of Apc initiated tumor formation by targeted inactivation of p21WAF1/cip1. We will pursue the p21 gene dosage effect on tumor formation and response to sulindac that we reported. We will determine if the wild-type gene in tumors from p21+/- mice is lost, mutated, or silenced by methylation, the level of expression of both p21 mRNA and protein in the tumors and intestinal mucosa, and how dietary and pharmacological modulation affect these levels.
Aim 2 extends the work on Apc/p21 to an Apc/p27 mouse model, to determine if effects we have seen involve overlapping or distinguishing mechanisms by which p21 and p27 act in cell cycling.
Aim 3 dissects the levels of expression and, where appropriate, activity of key components of the Apc-a-catenin-Tcf pathway, and linked components of the cell cycle machinery, in the intestinal mucosa, to distinguish among possible mechanisms by which p21 normally inhibits intestinal cell cycling in vivo, and through which its loss translates into elevated tumor formation.
Aim 4 extends the work to a new model we have made in which intestinal tumor formation is initiated by targeted inactivation of the Muc2 (mucin) gene. We will determine the mechanism of c-myc activation that takes place without alteration of a-catenin levels or intracellular localization, and address our hypothesis that in this model, diet interacts with inactivation of p21 in a manner, which differs from the Apc model. Finally, aim 5 will use surgical manipulation of portions of the intestinal tract of Apc+/- mice, assays of key molecules in signaling and cell cycling (including p21), and microarray analysis, to address the mechanisms by which the intestinal mucosa adapts when moved from a site of high probability of tumor formation, to one of low probability - and vice versa - and our hypothesis, based on our data, that this altered function will influence site-specific incidence of Apc initiated tumor formation.
