Clonal expansion of altered intestinal epithelial cells appears to precede frank transformation and tumorigenesis in the human colon. Mouse models indicate that this occurs through competitive advantages conferred to stem cells via somatic genetic and epigenetic alterations. Our objective is to determine the impact of Let-7 loss on stem cell activity and clonal expansion in the intestinal epithelium. We have discovered a very salient role for Let-7 in the regulation of a stem cell signature and a stem cell phenotype in the intestine via depletion of Let-7 miRNAs in the intestine epithelium. We achieved this in mouse models using a constitutive LIN28B transgene and early (late embryonic) Cre-Lox mediated deletion of the MirLet7c2/MirLet7b cluster for depleting Let-7 miRNAs. These mice also develop a highly penetrant tumor phenotype with the appearance of adenocarcinomas in the small intestine. However, we do not know the relative contribution of each of the 12 different Let-7 genes, and we do not know how precipitous loss in the adult may affect homeostasis of the intestinal epithelium. To address these questions we are interested in targeting Let-7 miRNAs in the adult intestinal epithelium via an inducible approach. Due to the large size of the Let-7 miRNA family (12 genes in 8 clusters, encoding 9 unique Let-7 miRNAs) multiple Let-7 genes may need to be inactivated in order to see a measureable phenotype. Alternatively, specific Let-7 genes may play a unique role. We propose a novel approach to address each of these quandaries. To explore the individual and combinatorial role of Let-7 miRNAs in controlling stem cell activity and clonal expansion we will target all of the clusters expressed in the intestinal epithelium via somatic mutagenesis. This will provide significant insight into the function of Let- miRNAs in restricting stem cell activity and clonal expansion. Our goal is also to develop a mosaic pattern of Let-7 mutations in a single animal, where 1 to 7 Let-7 clusters are mutated in various combinations throughout the intestine of a single animal. Through efforts in enteroids, we will also determine the relative effects on growth, proliferation, and colony forming potential through lentiviral-mediated expression of each of the 9 different Let-7 miRNA species. We will also examine how the growth repressive factors Apc, Pten, Smad4, and Trp53 affect expression of Let-7, where clonal expansion caused by their inactivation may depend, in part, on a failure to maintain sufficient levels of Let-7 miRNAs.
Stem cells are a multipotent population of cells in the intestine that can propagate mutations via clonal expansion, with some mutations conferring a survival advantage that precedes and promotes cellular carcinogenic transformation. We will investigate the role Let-7 microRNAs (miRNAs) have in repressing stem cell activity and clonal expansion in the intestinal epithelium via targeted somatic mutagenesis of multiple Let-7 clusters in genetically engineered mouse models. We will also investigate how these miRNAs repress colony forming potential (stem cell activity) in ex vivo cultures of intestinal epithelial organoid
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