MODULATION OF INTESTINAL HOMEOSTASIS AND CANCER

Targeting of key developmental pathways that regulate the normal differentiation and maturation of intestinal epithelial cells is a fundamental approach for inhibition of intestinal tumorigenesis. Important proof-of-principle has been published demonstrating that inhibition of Notch signaling in mice with a ¿-secretase inhibitor can drive intestinal carcinoma cells to differentiate both in vitro and in the ApcMin mouse, in the latter case causing tumor stasis and regression. We have developed a new mouse genetic model that targets Notch signaling specifically in both the small and large intestine by eliminating the enzyme that fucosylates the Notch receptor, a step that is necessary for interaction of Notch with its ligands. This produces a dramatic phenotype in the intestine: Notch signaling is down-regulated, and there is a tremendous expansion of the goblet, Paneth and enteroendocrine secretory cell lineages in both the colon and small intestine at 4 weeks of age, paralleled by a distortion of the proliferative compartment. These mice survive and appear healthy. However, by 9 months there is a very large inflammatory response in the intestinal mucosa of 100% of the mice, and evidence for development of dysplasia and adenoma formation. These data are the first demonstration of the importance of signaling specifically through Notch receptor-ligand interaction for intestinal mucosal homeostasis. Using this mouse, our overall goals are to determine how effective Notch signaling is in inhibition of intestinal tumor formation, what the longer-term consequences of this inhibition are for intestinal homeostasis, whether strategies that employ nutrients can modulate effects of Notch inhibition, and to understand the mechanisms by which Notch regulates intestinal homeostasis and its interaction with signals from the diet. In Aim 1, we will we will introduce the ApcMin allele into these mice to determine the efficacy of inhibition of tumor formation by the efficient inactivation of Notch and the longer term tumor suppression and changes in the mucosa caused by the down- regulation of Notch in the intestinal epithelial cells. Further, using a regulated villin-cre:er transgene, we will determine the kinetics with which mucosal lineage specific differentiation and the inflammatory response develop in these mice, and whether intestinal tumors recur with time, the efficacy of repeated rounds of Notch inactivation, and the longer term influence of this on the mucosa and health of the mice. Alternatives are described that utilize an Apc floxed mouse and the AOM model of colon tumorigenesis. In Aim 2, we will determine how the epithelial cell and the inflammatory cell responses in these mice evolve over 9 months, especially in terms of extent of inflammation, representation of major classes of inflammatory immune cells and elaboration of cytokines and other growth factors; how this is accelerated and amplified by a western-style diet that elevates risk for intestinal tumor formation; and how vitamin D supplementation, which is both chemopreventive and anti-inflammatory, can modulate these effects. Finally, Aim 3 will assay how macrophages isolated from control mice, from mice exhibiting inflammation, and from mice in which the inflammatory response has been modified by nutritional factors influence cell growth and differentiation of mouse intestinal epithelial cells both in vitro and when implanted under the kidney capsule in vivo. These experiments will determine the efficacy of down-regulation of Notch signaling in inhibiting intestinal tumor formation and the longer term effects of such down-regulation, how the responses to Notch down regulation are modulated by important nutritional factors that influence development of human colon cancer, and cellular and molecular mechanisms by which the inflammation of the intestine can be modulated by these same nutritional factors.