The intestinal epithelium has one of the highest turnovers of any tissue in the body. As such, intestinal epithelial self renewal is an important physiologic process which relies heavily on the stem cell. Progenitor or transit amplifying cells, arising from the stem cell ultimately reconstitute the differentiated cells of the intestinal epithelium. Due to the importance of these stem cells in self renewal, they must be protected from genotoxic insults such as radiation. RNA binding motif 3 (RBM3) is an RNA binding protein that is expressed in the intestinal epithelial cells. Prior work from the laboratory has shown that RBM3 upregulates several genes such as Cyclin D1, VEGF and COX-2 through RNA stability and translation. Furthermore, RBM3 had significant effects on cell proliferation, chemoresistance, radiation resistance, anchorage independent growth, resistance to mitotic catastrophe, and angiogenesis. Notch1 is a transmembrane signaling receptor. Upon activation, Notch1 is cleaved releasing the intracellular domain, which translocates to the nucleus and induces transcription of Notch-regulated genes. Several studies show that the Notch signaling pathway is a key player in regulating stem cell and progenitor cell hierarchy. Our preliminary data indicate that RBM3 can increase the number of cells expressing the putative stem cell marker DCLK1. My preliminary studies suggest that these cells have significant proliferative potential and show a tremendous increase in radiation resistance. In this proposal, we hypothesize that RBM3 increases the percentage of stem-like cells through altering the Notch signaling pathway and that these RBM3-induced stem-like cells have significant resistance to radiation.
In aim 1, we will validate DCLK1 and other putative stem cell markers as RBM3 induced factors that correlate with increased "stemness" within a cell population. We will use an inducible RBM3 overexpressing system and assay for changes in the expression of stem cell markers within intestinal epithelial cell lines. We will also determine that these cells display a stem like phenotype.
In aim 2, we will elucidate the role of RBM3 in affecting the Notch signaling cascade and characterize the interplay between the Notch signaling pathway and target stem cell markers. We plan to do this by using small molecule inhibitors of the Notch signaling pathway as well as siRNA-mediated knockdown of Notch pathway proteins. Finally in aim 3, we will determine the effect that RBM3 has on the stem cell phenotype in vivo. This will be performed by generating tumor xenografts using RBM3 overexpressing cells and RBM3 induced stem cells. The xenografts will be assayed for proliferation, ability to generate heterogenous tissue, and radiation resistance. These studies should reveal a mechanism by which RBM3 increases a stem-like phenotype within a heterogenous cell population and how these RBM3 induced stem cell phenotype generates resistance to radiation. We believe that these studies will be the first step in understanding the role for RBM3 in regulating intestinal stem cell physiology.
The intestinal stem cell is a key point in the self renewal pathway. Because the intestine is an organ most affected by exposure to radiation it is important to understand the stem cell physiology responsible for regenerating the intestine following an insult. The current study will further our understanding of how cells acquire and maintain a stem like phenotype and how this stem like phenotype can confer resistance to genotoxic insults such as radiation.