Molecular analysis of intestinal stem cells in the context of premature aging, chronic inflammation and colorectal carcinogenesis

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Colorectal cancer is one of the most common tumors in industrialized countries and associated with a high morbidity and mortality. Especially elderly patients and patients with inflammatory bowel disease have an increased risk to develop colorectal cancer. As cellular origin of tumorigenesis intestinal stem cells that express the so-called leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) recently moved into the scientific focus. The effects of aging and chronic inflammation on intestinal stem cells are still largely unknown and will be investigated in this research project.Animal models of chronic inflammation and premature aging will be combined with an Lgr5 model. Telomerase-deficient animals with dysfunctional telomeres will serve as an aging model. Animals after chemically-induced chronic colitis (DSS colitis) will serve as an inflammation model. Lgr5-positive intestinal stem cells are isolated from the animals of these models and respective control groups. Then the project will be divided into three parts: (I) The isolated intestinal stem cells, will be analyzed using next-generation sequencing (NGS) to identify genetic differences in tumor-associated signaling pathways. In this examination, prematurely-aged, chronically-inflamed and healthy stem cells will be compared. NGS analysis will be used to identify candidate genes, which have a positive or negative effect on growth, survival and proliferation of intestinal stem cells. (II) The next step will be the creation of specific shRNAs for these candidate genes and their effect on Lgr5-positive intestinal stem cells will be examined in vitro. Gene knockdowns, which lead to an increase in growth and survival of intestinal stem cells in vitro, will be used to produce genetically modified intestinal organoids. (III) These intestinal organoids will be transplanted into healthy animals in order to investigate the influence of aging and chronic inflammation on engraftment, differentiation and colorectal carcinogenesis in vivo.