Wnt/b-catenin signaling is a conserved developmental pathway that plays important roles in human disease. Mutations in adenomatous polyposis coli, a Wnt pathway component, are responsible for familial adenomatous polyposis syndrome and 80% of nonhereditary colorectal cancers. Since its identification two decades ago, however, the function of APC in Wnt signaling remains poorly understood. APC is part of a multi-protein complex that promotes ubiquitin-mediated degradation of the transcriptional coactivator, beta-catenin. Loss of APC function (due to truncating mutations or downregulating APC levels by RNAi) results in elevated b-catenin levels and ligand-independent activation of the Wnt pathway. We have developed a monoclonal antibody against LRP6, the Wnt coreceptor that inhibits Wnt3a-mediated activation of the Wnt pathway in cultured mammalian cells. Current models of Wnt signaling suggest that APC functions exclusively downstream of Wnt receptors. Surprisingly, our anti-LRP6 antibody (as well as LRP6 RNAi constructs) inhibits Wnt signaling in several cancer cell lines with mutation of APC as well as in cells depleted of APC by siRNA. Treatment of APC mutant cancer cells with the anti-LRP6 antibody downregulates intracellular levels of beta-catenin, consistent with effects on b-catenin degradation. In this proposal, we seek to uncover the link between APC and LRP6 in regulating Wnt pathway activation. We will assess whether loss of LRP6 function by anti-LRP6 antibody treatment or RNAi inhibits Wnt signaling in a larger panel of cancer lines with mutations in APC. We will test the possibility tha the Wnt pathway is activated at the level of the Wnt coreceptors (Frizzled and LRP6) upon loss of APC function. We will test whether other proteins upstream of the beta-catenin degradation complex are required for activation of the Wnt pathway upon APC loss of function by RNAi knockdown or expression of dominant-negative proteins. We predict that APC and LRP6 compete for binding to the beta-catenin degradation complex, and we will test this hypothesis in cultured cells, Xenopus egg extract, and with purified proteins. Finally, we propose to provide in vivo evidence using Xenopus embryos to confirm that the activation of Wnt target gene transcription in APC- morphant embryos can be blocked by LRP6 downregulation. These studies have the potential to provide insight into the function of an important tumor suppressor, APC, and to directly impact the development of therapeutics for the treatment of Wnt-driven diseases due to mutations in APC.
: This proposal focuses on a new mechanism of action for the human tumor suppressor, adenomatous polyposis coli protein (APC), in Wnt signaling, a major developmental and oncogenic pathway. We find that activation of the Wnt pathway due to APC mutation or loss of function is dependent on upstream components of the Wnt pathway and that APC regulates Wnt signalosome formation. We propose to perform biochemistry and Xenopus embryo studies to test our model that APC regulates signalosome formation via its interaction with the clathrin-mediated endocytic apparatus. Modified Project Summary: Wnt/?-catenin signaling is a conserved developmental pathway that plays important roles in human disease. Mutations in adenomatous polyposis coli (APC), a Wnt pathway component, are responsible for familial adenomatous polyposis syndrome and 80% of nonhereditary colorectal cancers. Since its identification two decades ago, however, the function of APC in Wnt signaling remains an enigma. APC is part of a multi- protein complex that promotes ubiquitin-mediated degradation of the transcriptional coactivator, ?-catenin. Loss of APC function (due to truncating mutations or downregulating APC levels by RNAi) results in elevated ?-catenin levels and ligand-independent activation of the Wnt pathway. Current models of Wnt signaling suggest that APC functions exclusively downstream of Wnt receptors. Paradoxically, we found that an anti- LRP6 antibody inhibits Wnt signaling in several cancer cell lines with mutation of APC as well as in cells depleted of APC by siRNA. Furthermore, treatment of APC-mutant cancer cells with an anti-LRP6 antibody downregulates intracellular levels of ?-catenin, consistent with activation of the Wnt pathway upstream of the ?- catenin complex in this genetic background. Based on these observations we originally proposed to study the interaction between APC and LRP6. Since our initial studies, we have found that Wnt pathway activation upon loss of APC function occurs at the level of both Wnt coreceptors (Frizzled and LRP6) and Dishevelled. In order to more precisely link loss of APC activity with activation of upstream membrane components, we now propose to take advantage of an inducible (auxin) APC degradation system and live-cell imaging to monitor activation of upstream components upon induction of APC degradation. Wnt signalosome formation is mediated by a clathrin-based endocytic process. We have found that dominant-negative dynamin and small molecule inhibitors of cathrin-mediated endocytosis have the capacity to block Wnt activation upon APC loss. Because inhibition of Wnt ligand secretion via small molecule inhibitors (porcupine inhibitors) does not block Wnt activation (with loss of APC) these studies suggest that APC regulates the endocytic process by inhibiting signalosome formation. We predict that APC interacts with components of the clathrin endocytic pathway to inhibit signalosome formation. We will test this hypothesis in cultured cells by performing biochemical and cell-based microscopic studies to determine the association between APC and known clathrin protein components. We have successfully expressed and purified recombinant version all of the major components of the ?-catenin degradation complex, including APC. Using this purified system, we will test whether ?-catenin degradation is influenced by components of the clathrin pathway. Finally, we propose to provide in vivo evidence using Xenopus embryos to confirm that activation of Wnt target gene transcription in APC-morphant embryos can be blocked by downregulation of LRP6 and Frizzled 7 and by injections of dominant-negative Dishevelled. MODIFIED SPECIFIC AIMS Wnt ligands are an evolutionarily conserved class of signaling molecules with critical roles in embryonic development and stem cell maintenance in adults. In the absence of Wnt ligand, the ?-catenin destruction complex (which includes the tumor suppressor adenomatous polyposis coli (APC) protein) targets ?-catenin for proteasomal degradation. Binding of Wnt ligand to the coreceptors Frizzled (Fz) and LDL receptor-related protein 5/6 (LRP5/6) stabilizes ?-catenin, whereupon it enters the nucleus to activate a Wnt-specific transcriptional program. Despite over two decades of investigation, the precise role of APC in Wnt signaling remains poorly understood. The prevailing model suggests that activation of Wnt signaling in APC-deficient cancer cells bypasses upstream membrane components of the pathway by directly inhibiting the ?-catenin degradation complex. Based on our original studies with the Wnt co-receptor, LRP6, and in a challenge to the current model, we proposed that activation of Wnt signaling due to loss of APC is dependent on LRP6, and LRP6 and APC compete for binding to the ?-catenin degradation complex. Upon further studies, we now present evidence indicating that Wnt signaling in APC-deficient cells requires activation of all the upstream pathway components in a manner independent of Wnt ligands. We now hypothesize that activation of Wnt signaling due to loss of APC is dependent on ligand-independent formation of Wnt signalosomes. We now propose a new model in which APC inhibits signalosome formation mediated by clathrin-directed endocytosis. We now propose the following modified aims: Aim 1. Determine how loss of APC activates Wnt signaling by modulating the composition and/or activity of Wnt receptor complexes at the plasma membrane. Our previous data demonstrated that inhibiting LRP6 function by mAb7E5 or LRP6 siRNA in several APC mutant colon cancer cell lines reduces ?- catenin levels and blocks Wnt signaling. Recently, we found that activation of the Wnt pathway in APC-mutant cells could be inhibited by dominant-negative Fz and dominant-negative Dishevelled, both acting upstream of LRP6. Activation of the Wnt receptor complex is not ligand dependent based on our findings that porcupine inhibitors (block Wnt secretion) and proteins that sequester Wnt ligands (sFRP2 and XWnt8-Fz5) do not inhibit Wnt pathway activation upon loss of APC function. Our preliminary studies using split-GFP technology indicate that LRP6 forms a complex with Fz and Axin upon APC knockdown. To more rigorously measure receptor activation in real-time upon loss of APC, we now propose to measure assembly of LRP6-Fz complexes and oligomerization of LRP6 via the use of fluorescence fluctuation spectroscopy in conjunction with auxin-induced degradation of endogenous APC. Aim 2. How does APC regulate clathrin-mediated signalosome formation (new aim)? Our recent studies demonstrate that Wnt pathway activation occurs upstream of LRP6 upon loss of APC function. However, activation is independent of Wnt ligands. Our new data indicate that blocking Wnt signalosome function via small molecule inhibitors of clathrin-mediated endocytosis or dominant-negative dynamin inhibits Wnt signaling in APC knockdown cells. These findings suggest that APC alters the earliest event in formation of a Wnt signalosome. Thus, we propose to perform biochemical studies involving co-immunoprecipitations and co- purification with clathrin-coated vesicles to determine whether APC interacts with components of the clathrin pathway. These studies will be complemented by co-immunolocalization studies between APC and clathrin- coated vesicle components. Finally, we will reconstitute the ?-catenin degradation complex using purified components and will test the effects of purified clathrin components on the interaction between APC and key components of the complex. Aim 3. Determine whether Wnt signaling upon APC removal is dependent on LRP6 (and upstream components) in a developing organism. The Xenopus laevis embryo affords a powerful, well-established system to study Wnt signaling in a natural tissue context of a vertebrate model organism. We will use whole embryos and explants to integrate our biochemical studies and explore their functional consequences at the cellular and organismal levels. Our data indicate that APC morpholino oligonucleotide knockdown stabilizes ?- catenin and promotes Wnt target gene transcription to a comparable degree as adding ligand alone, thereby demonstrating a similar role for APC in Xenopus and mammalian cells. In our original aim, we proposed to test the effects of knocking down LRP6 in APC morphants. We now expand this aim and propose to determine whether knocking down of upstream Wnt components (LRP6 and Fz7) or injection of dominant-negative Dishevelled promotes ?-catenin degradation and inhibit Wnt target gene expression in APC morphants.
Showing the most recent 10 out of 40 publications
