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 adenomatous polyposis coli protein (APC) in the Wnt signal transduction pathway. We find that activation of the Wnt pathway due to APC mutation or downregulation is dependent on the Wnt co-receptor, LRP6. We propose to perform biochemistry and Xenopus embryo studies to test our model that APC and LRP6 interact to regulate Wnt signaling.
|Chen, Tony W; Broadus, Matthew R; Huppert, Stacey S et al. (2014) Reconstitution Of ?-catenin degradation in Xenopus egg extract. J Vis Exp :|
|Hainline, Sarah G; Rickmyre, Jamie L; Neitzel, Leif R et al. (2014) The Drosophila MCPH1-B isoform is a substrate of the APCCdh1 E3 ubiquitin ligase complex. Biol Open 3:669-76|
|Murphy, Andrew J; Pierce, Janene; de Caestecker, Christian et al. (2014) CITED1 confers stemness to Wilms tumor and enhances tumorigenic responses when enriched in the nucleus. Oncotarget 5:386-402|
|Li, Bin; Fei, Dennis Liang; Flaveny, Colin A et al. (2014) Pyrvinium attenuates Hedgehog signaling downstream of smoothened. Cancer Res 74:4811-21|
|Wallace, Heather A; Merkle, Julie A; Yu, Michael C et al. (2014) TRIP/NOPO E3 ubiquitin ligase promotes ubiquitylation of DNA polymerase ?. Development 141:1332-41|
|Saito-Diaz, Kenyi; Chen, Tony W; Wang, Xiaoxi et al. (2013) The way Wnt works: components and mechanism. Growth Factors 31:1-31|
|Alfaro, Maria P; Deskins, Desirae L; Wallus, Meredith et al. (2013) A physiological role for connective tissue growth factor in early wound healing. Lab Invest 93:81-95|
|Tacchelly-Benites, Ofelia; Wang, Zhenghan; Yang, Eungi et al. (2013) Toggling a conformational switch in Wnt/?-catenin signaling: regulation of Axin phosphorylation. The phosphorylation state of Axin controls its scaffold function in two Wnt pathway protein complexes. Bioessays 35:1063-70|
|Barham, Whitney; Frump, Andrea L; Sherrill, Taylor P et al. (2013) Targeting the Wnt pathway in synovial sarcoma models. Cancer Discov 3:1286-301|
|Deskins, Desirae L; Bastakoty, Dikshya; Saraswati, Sarika et al. (2013) Human mesenchymal stromal cells: identifying assays to predict potency for therapeutic selection. Stem Cells Transl Med 2:151-8|
Showing the most recent 10 out of 22 publications