The Hippo (or alvador/Warts/Hippo, SWH) pathway is a well-conserved metazoan signaling cascade that restricts organ size in the fruit fly Drosophila melanogaster and limits tumorigenesis in mammals. The main target of this pathway is the Yorkie (Yki)/YAP1 transcriptional co-activator. Work from many labs studying Hippo and its target Yki/Yap1 in insect cells, human cells and mouse cells has coalesced around the hypothesis that the main regulators of this pathway are transmembrane molecules that mediate interactions between cells. According to this model, the main role of Yki/Yap1 is to serve as a key target of contact- inhibition mechanisms through which membrane-associated cell adhesion molecules 'sense'cell crowding and inhibit Yki/Yap1 to keep cells in a post-mitotic state. In our view this model is incomplete. As most epithelial cells spend their entire lives closely apposed with adjacent cells, large fluctuations in cell:cell adhesion seem to be an unlikely driver of the developmental growth that is normally dependent on Yki/Yap1. In unpublished work, we have found biochemical and genetic evidence of a novel form of adhesion-independent Yki regulation that plays a significant role in the physiologic growth of Drosophila tissues. We find that protein that mediate the cellular response to Ecdysone, the major steroid hormone in flies, are also required for cell:cell adhesion-independent modulation of Yki activity during normal, physiologic growth. Moreover, we can refine this Yki-regulatory effect to a molecular interaction between Yki itself and the Ec-responsive protein Taiman, whose human homolog Steroid Receptor Coactivator-3/Amplified-In-Breast Cancer-1 (SRC3/AIB-1) is amplified in a wide array of cancers. Our initial proteomic analysis of this Yorkie-Taiman complex has identified additional components that appear to define a macromolecular interaction network we have termed the Yorkie Nuclear Interactome (YNI). Our long-term goal is to understand how individual YNI proteins affect Yki-driven gene expression in Drosophila to identify mechanisms that trigger rearrangement of interactions within the YNI.
The aim of the current studies is to use cutting-edge proteomic techniques to analyze the macromolecular composition of the YNI in the whole organism and in the presence of Ec, and to couple this with genetic analyses of the role of individual YNI components in Yki nuclear activity in developing imaginal discs. In the first aim o this proposal, we will use quantitative affinity purification-mass spectrometry (AP-MS) technique to carry out in vivo identification of the larger suite of YNI proteins in the physiologic setting f imaginal disc cells, with an additional focus on identification of proteins that are specifically recruited to the YNI or displaced from it following exposure to elevated levels of circulating Ec. In the second aim, we will use the diverse array of genetic tools in Drosophila to assess the role of individual YNI proteins (identified in the AP- MS experiments) in controlling Yki activity in developing tissues.
The applicants have made a discovery in fruit flies that could directly impact how we treat hormone-driven diseases such as estrogen-dependent breast cancer and late-stage castration-resistant prostate cancer. At the heart of the discovery is an interaction between two key proteins, Yorkie and Taiman, which they find is key to hormone-driven growth in fruit flies. These proteins also exist in human cells, but were not known to regulate each other. The researchers have also discovered that other proteins are recruited into this Yorkie-Taiman complex, and propose to identify them in a long-term effort to understand and treat human cancers.