Although tissue stem cells have been proposed as candidates for cancer cells of origin, clear evidence demonstrating the transition of tissue stem cells to cancer cells is lacking for most tumors, and little is known of the mechanisms responsible for this transition. Indeed, a major obstacle in addressing these questions is an incomplete understanding of tissue stem cell physiology. Recent findings have identified basal epithelial cells that express the Sonic hedgehog (Shh) ligand in urinary bladder as urothelial stem cells, capable of regenerating the urothelium following injury in vivo and of forming bladder-like organoids from single cells in vitro. These findings moreover have identified a regulatory circuit, involving Hh/Wnt signal feedback between urothelial stem cells and stromal cells that controls regenerative response to injury by dramatically stimulating proliferation. The work proposed here makes use of injury-induced regeneration of the urinary bladder as a reliable means of initiating regenerative proliferation and differentiation, thus permitting a systematic study of tissue dynamics and signaling pathways involved in the regulation of normal urothelial stem cell physiology. These findings will then serve as a framework for identifying the bladder cancer cell of origin, and for elucidating the mechanisms underlying transition from normal stem cell to cancer cell. In Aim 1 of this research plan, the signaling pathways involved in control of proliferation and differentiation of bladder stem cells will be fully defined, using aser capture micro dissection and expression microarray analysis to identify new epithelial and stromal signaling activities during urothelial proliferation and differentiation. Based on preliminary studies, genetic analysis will be used to specifically test the role of stromally-produced FGF in promoting urothelial proliferation and of retinoic acid response in mediating differentiation. In Aim 2, genetic marking or ablation of basal stem cells or other urothelial cell types will be used in the setting of an enhanced method for induction of murine bladder cancer to trace the cancer cell of origin. A genetic model for bladder cancer induction will also be combined with marking of specific urothelial stem cells and other cell types to help assign the cancer cell of origin. In Aim 3, Hh and Wnt pathway activities will be manipulated genetically in epithelial or stromal cells during carcinogenesis to determine whether bladder cancer initiation or growth is affected. The transfer of human bladder tumors to new hosts with the manipulation of these pathways will reveal whether Hh and Wnt pathways affect growth or metastasis, and will provide new ways to improve prognosis of urothelial carcinoma. The results of these studies will have clear implications for the development of new therapeutic interventions for this malignant disease. In particular, the targeting of these pathways alone or in combination may block growth or induce differentiation of human bladder cancer cells, thus providing a strategy for the improvement of therapeutic options. These approaches may also provide a model for the attack of similar questions in other cancer types.