We have determined that intestinal tumor formation in mice with targeted inactivation of the cdk inhibitor p27kip1 is strictly a function of diet: very few tumors form in p27+/- or p27-/- mice fed chow diet, but tumor formation is significant in the hetero and homozygote null mice fed AIN76A, and for each genotype is greatly increased by a western style defined diet (high in fat and phosphate, low in Ca and vitamin D). Our overall hypothesis, based on work from our laboratory and that of others, is that alterations in the mechanisms of lineage specific differentiation, and more specifically, the differentiation of the goblet cell lineage, determine the probability of tumor initiation, and can be modulated by diet. The mechanisms of these nutritional-genetic interactions will be determined, and dissected along the crypt-lumen axis. We will use two mouse genetic models: the p27+/+, +/- and -/- mice;and a conditional knockout of Pofutl(F/F), the locus that encodes the fucosylatransferase required for activation of Notch signaling, since the state of activation of Notch signaling has now been demonstrated to be the determining factor in driving intestinal epithelial cells along either the absorptive or secretory cell pathways.
Aim 1 will determine whether elevating calcium and vitamin D in the western diet will reverse the increase in tumor formation the western diet promotes in the p27 model, as we and our collaborators have shown for other mouse genetic models, and will also provide tissue necessary for other aims.
Aim 2 will determine how the modulation of three key pathways that are determinants of intestinal cell maturation and lineage specific differentiation - Wnt signaling, Notch signaling and the machinery of cell cycle progression - are linked to the modulation of preneoplastic and neoplastic changes in the p27 model, and will be extended to determining how these changes are altered as a function of position along the crypt-villous axis, using the Weiser method that we have adapted to the mouse.
Aim 3 will use conditional, regulated inactivation of the Pofutl locus, to determine whether disruption of differentiation lineages in the intestinal tract directly leads to preneoplastic and neoplastic changes in the intestinal tract, and how these are modulated by diets that increase or decrease probability of tumor formation.
Aim 4 will use standard methodology, and a unique method of detection of active transcription sites in situ, to determine if gene signatures that we have already identified that characterize and distinguish intestinal cell lineages are perturbed as cells migrate along the crypt-lumen axis, in the p27 and Pofutl models;how diets that alter probability of tumor development modulate these perturbations;and, using microarray analysis, what other alterations in gene expression profiles characterize these affects. Interpretation of these data will be instructed by our extensive experience with these methods, novel statistical and bioinformatic methods, and our published and unpublished databases that dissect intestinal cell maturation pathways both in vitro and in vivo.
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