The canonical Wnt signaling pathway is required for the development of most organ systems and for the maintenance of stem cell populations in the adult. Aberrant signaling can drive the formation of tumors in several tissues. Axin proteins are considered negative regulators of the Wnt pathway due to their function in the destruction complex, which prevents signaling in the absence of Wnt ligand. Surprisingly, stabilization of Axin proteins by either genetic or pharmacological methods leads to an increase in signaling in the posterior of the e8.5 mouse embryo, whereas Wnt signaling is decreased in the head, suggesting that Wnt signaling is regulated in a tissue-specific manner. The goals of the proposed research are to understand how the positive and negative roles of Axin1 and Axin2 are tissue-specifically regulated during development. Specifically, the aims of this research are the characterization of the mechanisms controlling tissue-specific, Axin-stimulated Wnt signaling in the primitive streak, identification of the biochemical basis of Axin-stimulated Wnt signaling, and examination of whether stabilized Axin2 promotes tumorigenesis in the intestine and mammary gland. Using genetics and cell-based approaches, the proteins that establish Axin-stimulated Wnt signaling in the primitive streak will be identified. The role of high levels of Wnt ligand, the expression of core signaling components, the genetic interaction of potential modulators of the pathway, and intersecting signaling pathways expressed at this time and location in the mouse embryo will be examined for their contribution to Axin-stimulated Wnt signaling. These data will be used to recapitulate Axin-induced Wnt signaling in ES cells. To facilitate biochemical analysis of Axin-containing complexes and imaging of the cellular localization of Axin- containing complexes, a mouse that expresses a conditional tagged, stabilized Axin1 from the Rosa26 locus will be generated. Immunopurification of Axin-containing complexes from ES cells or tissues engaged in both Axin-inhibited and Axin-induced Wnt signaling will be assessed by Western blot to compare the binding of core pathway components. Mass spectrometry will be performed to identify whether unique proteins or post- translational modifications lead to different signaling outcomes in response to stabilized Axin. Finally, tissues in adults will be evaluated for increased Wnt signaling in response to stabilized Axin2, concentrating on populations maintained by Wnt-controlled stem cell niches. Additionally, the effect of stabilized Axin2 on two models of Wnt-related cancer, the Apcmin model of colorectal cancer and the MMTV-Wnt1 model of breast cancer, will be assessed, focusing on the incidence, onset, or invasiveness of the tumors that arise to determine whether some tumors increase in severity due to stabilized Axin proteins, addressing the utility of Axin-stabilizing drugs in cancer treatment.
The canonical Wnt signaling pathway is required for many aspects of embryonic development, is disrupted in a number of congenital human diseases, and is aberrantly regulated in many types of cancer. New evidence indicates that Axin proteins, generally considered to negatively regulate the Wnt pathway, stimulates the activity of the pathway in specific tissues at specific stages. The goal of the proposed research is to clarify the roles of Axin proteins in Wnt signaling, which is critical for understanding Wnt-related birth defects and to guide the development of effective cancer therapeutics.
