Altered expression of apicobasal polarity factors is associated with cancer in vertebrates and tissue overgrowth in model invertebrates, yet mechanisms by which these factors affect growth regulatory pathways are not well defined. We have previously identified the Drosophila melanogaster gene tsg101 (tumor susceptibility gene-101) as a factor that is required to simultaneously maintain tissue polarity and suppress tumor-like overgrowth of the larval imaginal discs, which are simple epithelial organs that give rise to most adult structures. We have shown that loss of tsg101 blocks endolysosomal turnover of the key apical membrane determinant Crumbs, and that this excess Crumbs protein accumulates at the apical membrane, spreads ectopically onto the basolateral membrane, and accumulates in late endosomes. As Crumbs nucleates an apical membrane- associated complex that interacts physically and functionally with the Par/aPKC and Scribble/Dlg polarity complexes, these defects in Crumbs regulation provide a ready explanation of the effect of tsg101 loss on epithelial polarity and architecture. Notably, over expressed Crumbs also drives substantial imaginal disc overgrowth, suggesting that this polarity factor has a second, more direct oncogenic role in tsg101 cells. In published work, we have found that the cytoplasmic tail of Crumbs contains a previously unrecognized growth-regulatory motif through which it interacts with and regulates the expanded protein, which is a key component of the conserved Hippo/Mst2 tumor suppressor pathway. These data show that Crumbs is a multi-functional protein capable of integrating junctional polarity signals with the well-established Hippo/Mst2 growth-regulatory pathway, and those lesions that disrupt Crumbs trafficking and localization may have very direct effects on the activity of this pathway. Our objectives in this proposal are to test the role of this newly discovered Crumbs-Hpo/Mst2 link in the excessive growth of tsg101 mutant tumors, and to identify novel components of the Crumbs-Hpo/Mst2 pathway from among a small collection of mutations we have identified as dominant-modifiers of Crumbs driven disc overgrowth. In particular, we will focus our analysis on the taiman gene, which encodes the fly homolog of the human Amplified in Breast Cancer-1 (AIB-1) oncogene, and that we believe acts as a transcriptional coactivator for the Crumbs-Hpo/Mst2 pathway. We will use standard genetic and molecular techniques in Drosophila organs and cultured cells to carry out the proposed studies.
Loss of the human gene Tumor Susceptibility Gene-101 (TSG101) has been suggested to promote formation of cancer, although mechanisms by which this might occur are not well understood. We have found that the fruit fly version of the TSG101 controls a protein called Crumbs, and that Crumbs in turn directly regulates the activity of a very well established cancer-regulatory pathway called the Hippo/Mst2 pathway. We propose to study this Crumbs-Hpo/Mst2 pathway in flies, and to determine how it contributes to the 'cancerous' growth of fly tumors and, by extension, human tumors.
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