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Open Full Page Published OnlineFirst June 29, 2015; DOI: 10.1158/1541-7786.MCR-15-0125 Oncogenes and Tumor Suppressors Molecular Cancer Research Structure, Dynamics, and Functionality of Tankyrase Inhibitor-Induced Degradasomes Tor Espen Thorvaldsen1,2, Nina Marie Pedersen1,2, Eva M. Wenzel1,2, Sebastian W. Schultz1,2, Andreas Brech1,2, Knut Liestøl1,3, Jo Waaler4, Stefan Krauss4, and Harald Stenmark1,2 Abstract Tankyrase (TNKS) enzymes, due to their poly(ADP-ribose) poly- revealed that the induced degradasomes in SW480 cells are mem- merase activity, have emerged as potential targets in experimental brane-free structures that consist of a filamentous assembly of high cancer therapy. However, the functional consequences of TNKS electron densities and discrete subdomains of various destruction inhibition remain incompletely resolved because of the binding complex components. Fluorescence recovery after photobleaching promiscuity of TNKS. One of the hallmarks of small-molecule experiments further demonstrated that b-catenin–mCherry was TNKS inhibitors (TNKSi) is the stabilization of AXIN, which plays rapidly turned over in the G007-LK-induced degradasomes, where- a pivotal role in the WNT/b-catenin signaling pathway. The present as GFP-TNKS1 remained stable. In conclusion, TNKS inhibition study focused on the known ability of TNKSi to induce cytoplasmic attenuates WNT/b-catenin signaling by promoting dynamic assem- puncta (degradasomes) consisting of components of the signal- blies of functional active destruction complexes into a TNKS-con- limiting WNT/b-catenin destruction complex. Using the colorectal taining scaffold even in the presence of an APC truncation. cancer cell line SW480 stably transfected with GFP-TNKS1, it was demonstrated that a TNKS-specific inhibitor (G007-LK) induces Implications: This study demonstrates that b-catenin is rapidly highly dynamic and mobile degradasomes that contain phosphor- turned over in highly dynamic assemblies of WNT destruction ylated b-catenin, ubiquitin, and b-TrCP. Likewise, G007-LK was complexes (degradasomes) upon tankyrase inhibition and pro- found to induce similar degradasomes in other colorectal cancer vides a direct mechanistic link between degradasome formation cell lines expressing wild-type or truncated versions of the degrada- and reduced WNT signaling in colorectal cancer cells. Mol Cancer some component APC. Super-resolution and electron microscopy Res; 13(11); 1487–501. Ó2015 AACR. Introduction regarded as the core complex components (5). In the WNT-off state, transcriptionally active b-catenin (ABC) levels are kept low The WNT/b-catenin signaling pathway plays a pivotal role in by CK1a/GSK3-mediated N-terminal phosphorylation of b-cate- fundamental biologic processes, including cell proliferation, cell nin and subsequent degradation by the ubiquitin-proteasome polarity, energy metabolism, and cell fate determination during system. Upon WNT activation, ABC escapes N-terminal phos- embryonic development and adult tissue homeostasis (1, 2). phorylation and proteasomal degradation, translocates to the Consequently, mutations in this pathway are linked to a broad nucleus, and initiates transcription of WNT/b-catenin-responsive range of human diseases, including cancer (3). The WNT/b-cate- genes by complexing predominantly with the TCF/LEF family of nin destruction complex regulates protein turnover of b-catenin, transcription factors (6). The mechanisms by which destruction the key mediator of canonical WNT signaling output (4). The complex activity is inhibited in the WNT-on state are currently structural proteins adenomatous polyposis coli (APC) and axis debated (7, 8). inhibition protein 1 and 2 (AXIN1/2), and the kinases casein The poly-ADP-ribosyltransferases tankyrase 1 (TNKS1) and kinase 1a (CK1a) and glycogen synthase kinase 3 (GSK3) are tankyrase 2 (TNKS2) modify acceptor proteins by transferring ADP-ribose moieties (poly-ADP-ribosylation) to amino acid side chains. Modified proteins are subsequently poly-ubiqui- 1 Centre for Cancer Biomedicine, Faculty of Medicine, Oslo University tinated and predominantly turned over by proteasomal degra- Hospital, Montebello, Oslo, Norway. 2Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Mon- dation (9). TNKS1/2 are involved in a wide range of cellular tebello, Oslo, Norway. 3Department of Informatics, University of Oslo, functions (10) and were recently shown to positively regulate 4 Oslo, Norway. Department of Microbiology, Unit for Cell Signaling, the WNT/b-catenin pathway through poly-ADP-ribosylation of Oslo University Hospital, Forskningsparken, Oslo, Norway. AXIN (11), the rate-limiting factor for destruction complex Note: Supplementary data for this article are available at Molecular Cancer stability and function (12). Implication of TNKS1/2 as drug- Research Online (http://mcr.aacrjournals.org/). gable targets in the WNT/b-catenin signaling pathway has Corresponding Author: Harald Stenmark, Institute for Cancer Research, The generated profound research on developing novel small-mol- Norwegian Radium Hospital, Montebello, N-0379 Oslo, Norway. Phone: 47- ecule inhibitors (9, 13, 14). Inhibition of the catalytic activity 22781818; Fax: 47-22781845; E-mail: [email protected] of TNKS1/2 reduces WNT/b-catenin signaling in both APC doi: 10.1158/1541-7786.MCR-15-0125 wild-type cells (e.g., in HEK293) and colorectal cancer cells Ó2015 American Association for Cancer Research. harboring APC truncations (e.g., in SW480; refs. 11, 15, 16). www.aacrjournals.org 1487 Downloaded from mcr.aacrjournals.org on September 30, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst June 29, 2015; DOI: 10.1158/1541-7786.MCR-15-0125 Thorvaldsen et al. Interestingly, immunofluorescence imaging of colorectal cancer Cell-based assays cells has revealed cytoplasmic puncta formation of destruction SW480, COLO320, and LS174T cell lines were purchased from complex components upon TNKSi treatment (15–17). Based the ATCC. Upon receipt, cells were frozen, and individual aliquots on the presence of phosphorylated b-catenin (phospho-b-cate- were taken into cell culture, typically for analysis within 15 nin [PBC]) in puncta, they have been designated functional passages. Cells were grown in RPMI (SW480 and COLO320) or degradasomes (18) that promote phosphorylation and subse- DMEM F12 (LS174T) medium supplemented with 10% (SW480 quent degradation of b-catenin. However, extensive structural and COLO320) or 15% (LS174T) FBS and 1% penicillin/strep- and functional studies on these central inhibitor-induced com- tomycin. Testing for mycoplasma contamination was performed plexes are lacking. every sixth week. For inhibition of TNKS activity, cells were treated G007-LK is a highly selective TNKSi that attenuates WNT- with 0.5 mmol/L G007-LK for 24 hours, unless specified otherwise. induced cell growth both in vivo and in vitro (19, 20). In the DMSO was used as a control. For inhibition of proteasomal present study, we used endogenous proteins and stably activity, cells were treated with 10 mmol/L MG132 for 1 hour, expressed fluorescent fusion proteins to investigate the molec- either alone or in combination with G007-LK. To generate a stable ular effects of G007-LK in APC-truncated SW480 cells. Key SW480 cell line expressing GFP-TNKS1, third-generation lenti- experiments were also reproduced in other colorectal cancer viral transduction was used as previously described (25). Detailed cell lines expressing wild-type or truncated versions of APC. We cloning procedures can be requested from the authors. Cells were examined the composition and structure of inhibitor-induced sorted by FACS and frozen. Individual aliquots were grown in cell degradasomes in SW480 cells by combining confocal, super- culture as described for SW480 cells. resolution, and electron microscopy. Moreover, dynamic prop- erties and functionality of the protein complexes were eluci- Western blot analysis dated by Western blotting, live-cell imaging, and quantitative Cells were rinsed in PBS and lysed in Laemmli lysis buffer [65.8 image analysis. Here, we show that TNKS inhibition by G007- mmol/L Tris-HCl, pH 6.8, 2.1% SDS, 26.3% (w/v) glycerol, LK induces highly mobile and structurally dynamic degrada- 0.01% bromophenol blue, dithiothreitol (DTT)]. Equal amounts somes. Importantly, there is a rapid turnover of b-catenin in the of whole cell lysate were separated by SDS-PAGE (Bio-Rad Lab- degradasomes as shown by photobleaching experiments. Fur- oratories) and blotted with polyvinylidene difluoride membranes thermore, high resolution microscopy enabled us for the first (Millipore). Immunodetection was performed with IRDye-con- time to reveal structural details of these complexes beyond the jugated secondary antibodies (LI-COR Biosciences). The Odyssey resolution limits of confocal microscopy. Our data give novel Imager system (LI-COR Biosciences) was used to scan all blots. insight into the mechanisms of TNKS inhibition and attenua- Protein bands were quantified using the Odyssey software. tion of WNT/b-catenin signaling in colorectal cancer cells and provide a direct mechanistic link between degradasome for- Confocal fluorescence microscopy mation and b-catenin degradation. Cells were grown on coverslips, fixed in paraformaldehyd, and further processed for antibody staining as previously described (26). Fluorescence signals were investigated with Zeiss LSM 710/ Materials and Methods 780 microscopes (Carl Zeiss MicroImaging GmbH) using stan-
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