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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 profound 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. 2015 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 2015 American Association for Cancer Research. harboring APC truncations (e.g., in SW480; refs. 11, 15, 16).

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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 properties 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- Antibodies, plasmids, and chemicals dard filter sets and laser lines and a Plan Apo 63 1.4NA oil lens. The following reagents were used: rabbit a-TNKS-1/2 (H-350, All images were taken at fixed intensity settings below saturation. specificity validated by colocalization with overexpressed GFP- TNKS1; Santa Cruz Biotechnology); rabbit a-AXIN1 (C95H11, Fluorescence recovery after photobleaching (FRAP) specificity validated by siRNA), rabbit a-AXIN2 (76G6, speci- experiments ficity validated by siRNA), rabbit a-APC, rabbit a-GSK-3b SW480 GFP-TNKS1 cells were seeded in 3.5 cm MatTek glass (27C10), rabbit a-PBC (phospho-Ser33/37/Thr41), rabbit bottom culture dishes, transfected with b-catenin–mCherry/Lipo- a-b-TrCP (specificity validated by colocalization with overex- fectamine 2000 transfection reagent, and treated with G007-LK pressed pcDNA3-Flag-b-TrCP; Cell Signaling Technology); for 24 hours. A Zeiss LSM 710 confocal microscope was used for mouse a-b-catenin (BD Transduction Laboratories); mouse photobleaching experiments. Culture dishes were kept in an a a -ABC (clone 8E7), mouse -Ubiquitin (FK1; Millipore); mouse incubation chamber (5% CO2,37C) during the time frame of a-b-ACTIN, mouse a-Flag (clone M2; Sigma-Aldrich); rabbit the experiment. b-catenin–mCherry was bleached with a 561-nm a-p62 (Progen); rabbit a-LC3 (Medical and Biological Labora- laser line and GFP-TNKS1 with a 405-nm laser line. Fluorescence tories); mouse a-LAMP1 (H4A3; Developmental Studies intensities were measured with the Zen software and data ana- Hybridoma Bank); rabbit anti-Hrs antiserum (21); human lyzed in the GraphPad Prism software. anti-EEA1 antiserum (gift from Ban-Hock Toh, Monash Univer- sity, Melbourne, Australia; ref. 22); Hoechst (Invitrogen/Dynal); Time-lapse live-cell imaging G007-LK (19); MG132 (Chalbiochem); Dimethyl sulphoxide SW480 GFP-TNKS1 cells were seeded in chambered coverglass (DMSO) (Sigma Aldrich); GFP-TNKS1 (provided by Sascha with 8 wells (Lab-Tek, Nunc). Unless specified otherwise, RPMI Beneke, University of Zurich, Switzerland); pcDNA3-Flag-bTrCP medium with 0.5 mmol/L G007-LK or DMSO (control) was added (Addgene, plasmid 10865; ref. 23); b-catenin-mCherry immediately before imaging of cells with the DeltaVision live-cell [generated from b-catenin-GFP (24) by standard molecular imaging system (Applied Precision, GE Healthcare). Washout of biology methods]; secondary antibodies for immunofluores- inhibitor-medium was performed without removal of the imaging cence imaging (Jackson ImmunoResearch Laboratories and dish from the microscope incubation chamber (5% CO2,37C) Molecular Probes); secondary antibodies for Western blot and imaging was resumed immediately after replacement of medi- analysis (IRDye, Li-Cor Biosciences). um. Movies were processed and analyzed in the ImageJ/Fiji

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software (27). A customized Fiji script written in Jython was used to S1). Initially, puncta were numerous, small-sized, and barely measure the fluorescence intensity of degradasomes over time (28). detectable. These puncta were widespread in the cytoplasm and showed high mobility. However, over a time frame of 7 hours, ScanR high-throughput microscopy they appeared to coalesce, generating enlarged structures with Cells were grown on coverslips and further processed for perinuclear localization. Of note, puncta mobility gradually antibody staining as described for confocal microscopy samples. declined with increasing size. Images were automatically taken using the Olympus ScanR sys- To further elucidate the composition of TNKS1 containing tem with an UPLSAPO 40/0.95 objective. All images were taken puncta, SW480 cells were treated with G007-LK for 24 hours with the same settings and below pixel saturation. The Olympus before fixation and immunostaining with antibodies against ScanR analysis program was used to detect and measure intensity known destruction complex constituents. Confocal fluorescence of GFP-TNKS1, AXIN2, and PBC in G007-LK-induced degrada- microscopy showed AXIN1, AXIN2, GSK3b, TNKS1/2, and APC somes. Typically between 400 and 500 cells were analyzed per colocalizing with b-catenin in G007-LK-induced puncta (Fig. 2A), condition in each experiment. indicating that these structures contain the main components of the WNT destruction complex. Likewise, we observed colocaliza- Statistics tion of TNKS with endogenous destruction complex components Testing of the different quantifications was done by using mixed in SW480 cells stably expressing GFP-TNKS1 upon treatment with effect models (experiments as random factor) for Fig. 4D and for G007-LK (Supplementary Fig. S1A) but not in SW480 cells treated Fig. 5A, in the latter case using Tukey's Honest Significance Difference with DMSO alone (Fig. 2A). Of note, the inhibitor-induced puncta (HSD) for handling multiple comparisons. The testing was done on contained both ABC and PBC (Supplementary Fig. S1B). Key log-transformed values and two-sided P values are given in the figure experiments were also reproduced with a different chemotype of legends.ForFig.5C,thedataweretestedwithaone-samplet test. TNKSi (XAV939; ref. 11). SW480 cells expressing GFP-TNKS1 were treated with XAV939 for 24 hours and stained with antibodies Other materials and methods against b-catenin and Axin2. As expected, XAV939-induced puncta See Supplementary Materials and Methods. contained all three proteins (Supplementary Fig. S1C). To examine any possible association with endocytic and autophagic pathways, we checked localization of markers of these Results pathways in G007-LK-treated SW480 cells. However, G007-LK- G007-LK induces mobile degradasomes and degradation of induced puncta did neither colocalize with markers of early b-catenin in SW480 colorectal cancer cells endosomes (EEA1), multivesicular endosomes (HRS), and late The colorectal cancer cell line SW480 exhibits high cellular levels endosomes/lysosomes (LAMP1) nor with autophagosomal of b-catenin due to a mutation in the APC gene and is frequently (LC3/p62) markers that we tested (Fig. 2B and Supplementary used as a model for WNT-dependent tumors (29). Recent studies Fig. S2). This is consistent with the idea that G007-LK-induced have reported AXIN puncta formation and reduced b-catenin levels puncta represent proteninaceous, membrane-free structures rath- in SW480 cells upon treatment with different TNKSi (15–17). er than vesicular compartments. Taken together, we conclude that Consistent with these observations, fluorescence imaging revealed G007-LK treatment of SW480 cells induces accumulation of high nuclear b-catenin levels (antibody detecting total b-catenin, destruction complex components in mobile cytoplasmic puncta hereafter referred to as b-catenin) and low AXIN2 expression in (hereafter referred to as degradasomes), accompanied by a sub- DMSO-treated SW480 cells (Fig. 1A, top). In contrast, G007-LK stantial reduction in ABC. treatment induced accumulation of b-catenin in cytoplasmic AXIN2-positive puncta, coinciding with reduced nuclear b-catenin. High resolution imaging reveals novel structural details of Puncta were distributed throughout the cytoplasm and varied in degradasomes size with the biggest puncta measuring up to approximately 1 mm In order to investigate the structure of the G007-LK-induced in diameter (Fig. 1A, bottom). Western blot analysis confirmed an degradasomes at higher resolution, we utilized structured illumi- inverse correlation between G007-LK incubation time and b-cate- nation microscopy on the SW480 cells stably expressing GFP- nin levels, with a fraction of the b-catenin pool being resistant to TNKS1 that were treated with G007-LK for 24 hours. Although the degradation. However, detecting the levels of ABC with an anti- degradasomes appeared more or less perfectly round and homo- body specificforb-catenin dephosphorylated on Ser37 and Thr41 geneous in conventional confocal microscopy, super-resolution revealed a drastic decline of ABC over time. Indeed, after 24 hours microscopy revealed a more complex substructure (Fig. 3A and B): of G007-LK treatment hardly any ABC was present. In contrast, the GFP-TNKS, b-catenin and AXIN2 appeared to form intertwined amount of AXIN2 was substantially increased, accompanied by a meshworks without showing a particular colocalization between moderate increase in AXIN1 levels (Fig. 1B). any two of these components. To investigate the induction of puncta in more detail we To investigate the degradasomes at the ultrastructural level, we performed live imaging of G007-LK-treated SW480 cells stably treated SW480 cells stably expressing GFP-TNKS1 for 24 hours expressing GFP-TNKS1 (Fig. 1C), as TNKS1 localizes to inhibitor- with G007-LK and examined the induced degradasomes by cor- induced puncta (Fig. 2A). We chose to take advantage of a stable relative light and electron microscopy (Fig. 3C–F). Images of cell line generated by lentiviral transduction in which the expres- degradasomes from confocal microscopy (Fig. 3C) and electron sion of GFP-TNKS1 is under the control of a weak phosphoglyc- micrographs of the same cell (Fig. 3D) were superimposed, which erate kinase 1 promoter in order to ensure even and low expres- revealed the fibrillar nature of the degradasome structures and the sion levels of the fluorescent fusion protein (30). Live-cell imaging lack of surrounding membranes (Fig. 3E). Higher magnification revealed a rapid induction of GFP-TNKS1-positive puncta after revealed that the degradasome consists of a filamentous assembly G007-LK addition (<60 minutes; Fig. 1C, Supplementary Movie of high electron densities, possibly interspersed by cytosol (Fig.

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Figure 1. Induction of AXIN puncta and degradation of b-catenin in G007-LK-treated SW480 cells. A, confocal sections through SW480 cells treated with DMSO (top) or G007-LK (bottom) for 24 hours and immunostained with antibodies against AXIN2 and b-catenin. Control cells show high nuclear levels of b-catenin and low AXIN2 expression. Treatment with G007-LK increases AXIN2 levels and causes a substantial reduction in the amount of nuclear b-catenin. Magniﬁcation of G007-LK-treated cells shows colocalization of AXIN2 (green) with b-catenin (red) in cytoplasmic puncta. Blue, Hoechst. Scale bar, 10 mm. B, Western blots of whole cell lysates from SW480 cells treated with G007-LK for 0, 6, 24, and 48 hours, respectively. Total and active b-catenin levels are progressively reduced upon G007-LK treatment, coincident with increased AXIN1/2 levels. Equal protein loading is documented by staining of b-ACTIN. C, stably GFP-TNKS1 expressing SW480 cells examined by DeltaVision live microscopy immediately after adding G007-LK (Supplementary Movie S1). Images captured every fourth minute during a time frame of 7 hours reveal a rapid induction of GFP-TNKS1-positive puncta. Of note, we observe a distinct localization of GFP-TNKS1 to the spindle poles during mitosis, which is in line with the established role of TNKS in centrosome-related processes (13). Still frames of representative cells are shown. Minutes after adding G007-LK are indicated.

3F). In addition, we found membranes in close proximity to the membranes, which is in line with the lack of autophagic markers degradasomes. As we had collected 55-nm thick serial sections we (LC3 and p62) as revealed with immunoﬂuorescence (Fig. 2B and were able to obtain a 3D model (Fig. 3G) based on 10 consecutive Supplementary Fig. S2). sections (Supplementary Fig. S3). Following the membranes in 3D it became clear that the membranes identiﬁed in each single G007-LK-induced degradasomes are rapidly dissolved upon section (Fig. 3E) are connected with each other and are part of the inhibitor-washout endoplasmic reticulum (ER). However, there is no indication that Live microscopy revealed a rapid induction of degradasomes these membranes are in direct contact with the degradasome and upon TNKSi treatment (Fig. 1C, Supplementary Movie S1). Because we can exclude that the degradasome is surrounded by autophagic the WNT destruction complex is believed to represent a dynamic

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Figure 2. WNT destruction complex components colocalize in G007-LK-induced degradasomes. Confocal sections through SW480 cells treated with either DMSO or G007-LK for 24 hours and immunostained with antibodies as indicated. A, merged images show b-catenin (red) colocalizing with destruction complex components (green) in G007-LK-induced cytoplasmic puncta (right). All destruction complex components show diffuse cytoplasmic staining in control cells (left). Scale bar, 2 mm. B, endosomal and autophagosomal markers (green) do not colocalize with b-catenin (red) in cytoplasmic puncta, as shown in merged images (right). Scale bar, 2 mm. Blue, Hoechst. EEA1, early endosome antigen 1; p62, ubiquitin-binding protein p62.

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Figure 3. High resolution imaging of G007-LK-induced degradasomes. G007-LK-induced degradasomes contain a substructure and are in close proximity, but not directly connected, to ER membrane sheets. A, SW480 cells stably expressing GFP-TNKS1 treated with G007-LK for 24 hours and stained against total b-catenin and AXIN2. 3D SIM imaging reveals an irregular shape of the induced degradasomes and a nonhomogeneous distribution of GFP-TNKS1 (green), b-catenin (red), and AXIN2 (blue) in subdomains. Displayed is a maximum intensity projection. Scale bar, 1 mm. Individual complexes were enlarged for better visualization (1.2 1.2 mm). White, Hoechst. B, 3D reconstruction of the same field of view (as in A) for better visualization. Note the irregular shape and the subdomain structure within the degradasomes. C, confocal section of SW480 cells stably expressing GFP-TNKS1 treated with G007-LK for 24 hours. Blue, Hoechst. D, low-magnification EM of the cells from C. The section (þ275 nm) is part of a series of 10 consecutive, 55-nm thick sections that span over the whole degradasome. E, EM of the boxed area in D. The outline of ER membranes that are in close proximity to the cluster are displayed in green, and the substructure in the cluster is highlighted in pink. F, high magnification EM of the boxed area in E. Note the electrondense, granulated substructure and the absence of discernible membranes within the cluster. The electrondense substructure is interspersed with less electrondense areas that are not distinguishable from the surrounding cytosol. G, 3D model of the substructure of the cluster (pink) and the surrounding ER membranes (green). The 3D model is based on the outlines of the respective features in the 10 consecutive EM sections (as represented in E).

multiprotein assembly (31), we next tested the stability of the GFP-TNKS1 were treated with G007-LK for 4 hours (Fig. 4A, inhibitor-induced puncta in the absence of TNKS inhibition by Supplementary Movie S2). Next, cells were washed and the inhib- inhibitor washout experiments. First, SW480 cells expressing itor-containing medium was replaced with regular RPMI medium.

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Images were captured every minute for 1 hour following washout ylation of b-catenin and subsequent degradation by the ubi- with a DeltaVision fluorescence live microscope. Strikingly, the quitin-proteasome system. To elucidate the functionality of induced degradasomes disappeared gradually in the absence of G007-LK-induced degradasomes in SW480 cells, we decided to TNKS inhibition and were hardly detectable after 1 hour (Fig. 4A, investigate phosphorylation of b-catenin within the com- Supplementary Movie S2). Moreover, we were able to reinduce plexes. Initially, we performed Western blot analysis on complex formation by treating the cells with G007-LK for another SW480 cells incubated with G007-LK for 24 hours and immu- 22 hours (Supplementary Fig. S4A). noblotted with an antibody specifically detecting PBC (Fig. 5A, To compare the kinetics of TNKS and b-catenin in degrada- lane 2). As b-catenin has been shown to be degraded by somes after washout of the inhibitor, we transfected GFP-TNKS1 proteasomes, the proteasomal inhibitor MG132 was added to expressing cells with b-catenin–mCherry and incubated with G007-LK-containing medium 1 hour before lysis. Inhibition of G007-LK for 24 hours. Images were then captured with the proteasomal activity showed a substantial increase in PBC DeltaVision microscope every minute for 1 hour following either protein levels, indicating a high rate of b-catenin turnover in washout of, or continued treatment (control) with, the inhibitor G007-LK-treated cells (Fig. 5A, lane 3). We also observed a (Fig. 4B, Supplementary Movie S3). We measured the intensity of minor increase in PBC levels with MG132 treatment alone GFP and mCherry in individual degradasomes (N ¼ 12 [control] (Fig. 5A, lane 4), which is in accordance with a modest þ 12 [washout]) at each time point. Quantitative analyses showed increase in AXIN levels (data not shown) and previously that the fluorescence intensity did not change during 1-hour published observations (32, 33). Protein levels were quantified continued treatment with G007-LK. However, after washout of using the Odyssey imaging software and normalized to the inhibitor, the GFP and mCherry signals declined in parallel, b-ACTIN (Fig. 5A, graph). revealing similar dissociation kinetics for TNKS and b-catenin. We next immunostained G007-LK-treated SW480 cells Next, to further characterize the stability of destruction complex expressing GFP-TNKS1 with the antibody specifically detecting components within the degradasomes, GFP-TNKS1 expressing PBC targeted for proteasomal degradation. Confocal micros- SW480 cells were cultured on coverslips and treated with G007-LK copy showed that PBC localizes to inhibitor-induced degrada- for 24 hours. Individual samples were fixed at 24 hours G007-LK somes (Supplementary Fig. S5A). Based on this, we reasoned treatment and after 15 and 60 minutes washout with inhibitor- that proteasomal inhibition would allow PBC to accumulate in free medium, respectively. All samples were stained for endoge- the complexes. The proteasomal inhibitor MG132 was there- nous AXIN2 and b-catenin. Fluorescence microscopy revealed a fore added to G007-LK-containing medium 1 hour before cells rapid decline in puncta size and fluorescence intensity for all the were fixed and stained. Indeed, combining MG132 with G007- three tested complex components (Supplementary Fig. S4B). LK resulted in distinct accumulation of PBC, and an increased While b-catenin seemed to redistribute in the cytoplasm and to colocalization of PBC and GFP-TNKS1 was observed (Supple- the nucleus, the GFP-TNKS1 and AXIN2 signal completely dis- mentary Fig. S5A). The observed effect of proteasomal inhibi- appeared within 1 hour washout. tion was quantified after high-throughput image acquisition Our next question was whether the degradasome simply dis- using an Olympus ScanR microscope. The intensity of PBC solves in the cytoplasm or if the participating proteins are degrad- within GFP-TNKS1 puncta was quantified using the ScanR ed when the TNKSi is removed. To address this, we performed analysis software, and quantitative analysis of fluorescence Western blot analysis of SW480 GFP-TNKS1 cells to examine intensity displayed a 2.5-fold increase in degradasome-associ- protein levels of central degradasome components (TNKS, ated PBC when including MG132 the last hour of G007-LK- AXIN2, and b-catenin) upon washout of G007-LK. Individual treatment (Fig. 5B and C). In contrast, the fluorescence intensity samples were lysed and scraped in sample buffer at 24 hours of of GFP-TNKS1 did not change during incubation with G007-LK treatment and up to 90 minutes after washout of the inhibitor. and MG132 together. Protein levels were quantified using the Odyssey imaging software Ubiquitination is a prerequisite for proteasomal degradation and normalized to b-ACTIN. AXIN2 and TNKS protein levels of b-catenin (34). To examine whether ubiquitin conjugates decreased immediately after washout of G007-LK, although there colocalize with degradasomes, we immunostained G007-LK- was no significant change in the amount of total b-catenin within treated GFP-TNKS1-expressing cells with an antibody detecting the measured time interval (Fig. 4C and D). AXIN is regarded as poly-ubiquitin conjugates. Confocal microscopy revealed ubi- the rate-limiting factor for destruction complex stability (12) and quitin-specific staining colocalizing with GFP-TNKS1 (Supple- the rapid degradation of AXIN2 may explain the dissipation of mentaryFig.S5B,top). puncta observed by microscopy. We also noticed a substantial Based on the presence of ubiquitin, we further stained GFP- reduction in PBC levels after washout of G007-LK, indicating TNKS1 expressing SW480 cells with an antibody specifically decreased phosphorylation of b-catenin by the destruction detecting b-transducing-repeat-containing protein (b-TrCP). complex (Supplementary Fig. S4C). Indeed, ABC levels were b-TrCP is a component of the E3 ubiquitin ligase responsible for completely rescued upon prolonged inhibitor-washout (Supple- ubiquitination of b-catenin in the WNT-off state (34). Indeed, mentary Fig. S4D). Taken together, our results show that TNKSi- confocal microscopy showed a distinct colocalization of endog- induced degradasomes are extremely dynamic and tightly regu- enous b-TrCP or overexpressed Flag-b-TrCP with GFP-TNKS1 in lated in terms of their assembly and disassembly. the G007-LK-induced degradasomes (Supplementary Fig. S5B, bottom, and S5C). Furthermore, multiple-color fluorescence G007-LK-induced degradasomes in SW480 cells represent imaging of inhibitor-treated cells revealed colocalization between functional assemblies of destruction complex components GFP-TNKS1, b-TrCP, and Ubiquitin (Fig. 5D). From these data targeting b-catenin for proteasomal degradation we propose that G007-LK-induced degradasomes in SW480 cells In cells with functional destruction complexes, ABC levels are functional by targeting b-catenin for proteasomal degrad- are kept low by CK1a/GSK3-mediated N-terminal phosphor- ation through phosphorylation and ubiquitination.

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Figure 4. G007-LK-induced degradasomes are rapidly dissolved upon inhibitor washout. A, A0: SW480 GFP-TNKS1 cells were examined with DeltaVision live microscopy after adding G007-LK. Images were captured every second minute during a time frame of 4 hours. (Continued on the following page.)

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TNKSi-induced degradasomes in additional colorectal cancer microscope, which revealed a distinct puncta formation. Indi- cell lines with wild-type or truncated APC vidual b-catenin–mCherry puncta (N ¼ 32) were photo- Interestingly, TNKSi-induced AXIN puncta have also been bleached (561 nm laser) to 35% of starting fluorescence and observed in cells expressing a more severe APC truncation at recovered to 76% of prebleach intensity within approximately 1 amino acid 811 that lacks b-catenin and AXIN-binding sites 1minute(T/2: 13.74 s 1.14 SEM). Despite the rapid flow of (COLO320 cells; ref. 17). However, these have been termed b-catenin into puncta, fluorescence intensity of unbleached "stalled degradasomes" due to their inability to promote reduced neighboring puncta did not increase during the time frame of ABC levels in cells treated with the TNKSi XAV939. We noticed these experiments, indicating a concomitant constant outflow distinct G007-LK-induced AXIN2 puncta in the COLO320 cells, of b-catenin (data not shown). GFP-TNKS1 in the same puncta which colocalized with b-catenin, PBC, and GSK3b, but not with was photobleached (405 nm laser) to 10% of starting fluo- APC (Fig. 6A, Supplementary Fig. S6A). In contrast to the findings rescence. However, the GFP signal did not recover within of de la Roche and colleagues, our Western blot analysis revealed a approximately 1 minute after bleaching (Fig. 7A and B, Sup- substantial reduction in ABC levels after G007-LK treatment, plementary Fig. S7). TNKS therefore seems to be considerably accompanied by increased levels of AXIN1/2 (Supplementary more stable than b-catenin in G007-LK-induced puncta, where- Fig. S6B). In line with our findings, Lau and colleagues recently as b-catenin is constantly turned over. showed strong inhibition of WNT/-b-catenin signaling at the level of Wnt target gene transcription in COLO320 cells treated with G007-LK (20). In future experiments we plan to further Discussion characterize the components and function of degradasomes in Extensive research has generated a detailed understanding of COLO320 cells. the WNT signaling pathway and its molecular aberrations that We next investigated the effect of G007-LK in the APC wild-type underlie cancer development (3). Mutations in the APC gene are colorectal cancer cell line LS174T. In these cells, b-catenin is found in the majority of colorectal cancer cells (37). Despite this, homozygously mutated at Ser45 and therefore cannot be phos- no targeted therapeutics for APC-mutant colorectal cancer cells phorylated by CK1 and consequently GSK3 (35). Confocal have advanced to clinical testing (38). TNKS1/2 were discovered microscopy showed AXIN2 puncta colocalizing with b-catenin, as targets of WNT pathway inhibition in 2009 (11) and several GSK3, and APC in cells treated with G007-LK (Fig. 6B). Moreover, small-molecule TNKSi have been identified in the following years we observed a substantial increase in AXIN1/2 levels by Western (15, 16, 19, 39–41). TNKSi have been proposed to promote blotting (Supplementary Fig. S6C). The levels of ABC did not b-catenin degradation through stabilization of AXIN, the concen- change upon TNKS inhibition, which was expected due to the tration limiting factor for destruction complex stability (12). b-catenin stabilizing Ser45 mutation in LS174T cells. Our results However, studies showing a mechanistic link between AXIN show that induction of degradasomes upon TNKS inhibition also protein stabilization and b-catenin degradation are lacking. Inter- pertains to APC wild-type cell lines. estingly, immunofluorescence imaging of colorectal cancer cells has recently described puncta of colocalized destruction complex b-catenin is constantly turned over in degradasomes components (degradasomes) upon TNKSi treatment (15–17). FRAP can be used to investigate protein dynamics and activity Although it is well established that exogenous AXIN puncta in living cells. Fluorescent molecules are irreversibly photo- are induced upon AXIN overexpression, TNKSi have enabled bleached in a small area of the cell and subsequent diffusion visualization and characterization of endogenous degrada- of surrounding nonbleached fluorescent molecules into the somes. To elucidate the role of these complexes in reducing bleached area can be recorded (36). The purpose of including aberrant WNT signaling, we focused on APC-mutated SW480 FRAP in our study was to investigate the kinetics of exchange cells treated with the TNKSi G007-LK, but verified our key results between punctuate (i.e., degradasome-associated) and diffuse in two other cell lines (COLO320 and LS174T) and with a cytoplasmic b-catenin during G007-LK-treatment. The SW480 different TNKSi (XAV939). G007-LK is a potent and selective cells stably expressing GFP-TNKS1 were transfected with b-cate- TNKSi, which has shown antitumor efficacy in xenograft and nin–mCherry. Transfection medium was replaced after 5 hours, genetically engineered mouse colorectal cancer models (20). In followed by 24-hour incubation with G007-LK medium. Trans- accordance with recent reports, the levels of b-catenin were greatly fected cells were investigated with a Zeiss LSM 710 confocal reduced upon inhibitor treatment, accompanied by increased

(Continued.) Please note that due to a short delay between addition of inhibitor and start of image acquisition, small GFP-TNKS1 puncta are already visible in the first frame (00). This underlines the rapid induction of degradasomes upon G007-LK-treatment. A00: inhibitor medium was replaced with regular RPMI medium and images were captured every minute during a time frame of 1 hour. GFP-TNKS1 puncta disappear rapidly upon washout of inhibitor. Still frames (Supplementary Movie S2) of the representative cells are shown. Scale bar, 10 mm. B, SW480 GFP-TNKS1 cells coexpressing b-catenin–mCherry were incubated with G007-LK for 24 hours to allow for degradasome formation. The inhibitor was removed (washout) or not (control) directly before the start of image acquisition. Images were captured every minute during a time frame of 1 hour. Still frames (Supplementary Movie S3) of representative cells (top) and clusters (bottom) are shown. Fluorescence intensities of GFP and mCherry signals were quantified from individual degradasomes (at least 12 clusters from 3 independent experiments per condition), normalized and averaged (SD). Scale bar, 10 mm. C, SW480 GFP-TNKS1 cells were seeded in 5-cm dishes and incubated with G007-LK for 24 hours. Cells were washed with PBS and either lysed for Western blotting or further incubated with regular RPMI medium for the given time point and then lysed. Inthefigure, time point 00 refers to 24-hour incubation with G007-LK and is used as point 0 for washout quantifications. Immunoblotting was performed with antibodies against TNKS1, AXIN2, and b-catenin (total). Asterisk: splice variant of TNKS1. b-ACTIN was used as a loading control. One representative blot is shown. D, quantification of Western blots from C. Six independent experiments (SD) were used for quantification using Licor Odyssey software. Twenty-four hours of incubation with G007-LK was used as point 00 for the washout experiment. Its values were set to 1 and values from the independent time points are shown relative to this. Differences over time were tested using a mixed effect model (experiments as random factor). TNKS1 and AXIN2 show a significant reduction in protein levels (P < 0.0001) from time 00 to 900, in contrast to b-catenin, which did not change significantly after washout.

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Figure 5. Degradasomes target b-catenin for proteasomal degradation. A, SW480 cells were incubated with DMSO or G007-LK for 24 hours, for the last hour MG132 was added to one sample treated with DMSO and one with G007-LK. Cells were then lysed and whole cell lysate was applied for Western blotting. Membranes were incubated with antibodies against PBC and b-ACTIN (loading control). One representative blot is shown and the graph shows quantiﬁcation of three independent experiments (SD). The bars represent PBC related to b-ACTIN. (Continued on the following page.)

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AXIN levels and induction of degradasomes. High resolution containing protein b-TrCP (substrate recognition subunit for the imaging techniques were applied for obtaining novel structural SCF-TrCP E3 ubiquitin ligases) is disrupted (8). This abrogates details of the induced degradasomes and revealed a striking ubiquitination of b-catenin and saturates the destruction complex substructure with discrete subdomains of various destruction with PBC. Newly synthesized b-catenin thus escapes destruction complex components. Moreover, live-cell imaging displayed rap- and initiates transcription of WNT responsive genes. Functionality id induction of degradasomes upon TNKSi treatment and their of TNKSi-induced degradasomes has previously been based on immediate resolution after inhibitor washout. Importantly, quan- colocalization with PBC and increased PBC protein levels on titative image analyses showed that the G007-LK-induced com- Western blot analysis (16, 17). However, based on the model of plexes represent active sites of b-catenin processing, thus provid- Li and colleagues, these experiments do not prove degradational ing a direct mechanistic link between degradasome formation and activity in the complex per se. By implementing high-throughput enhanced b-catenin degradation in TNKSi-treated colorectal can- image acquisition (ScanR microscope), we quantitatively showed cer cells. that the PBC accumulated in degradasomes when combining Our live-cell imaging approach permitted investigation of TNKSi and proteasomal inhibitors. This was evident by an TNKSi-induced degradasomes dynamics for the first time. We increased fluorescence intensity and size of the PBC positive observed a strikingly rapid induction of degradasomes (<60 puncta. Furthermore, confocal imaging revealed colocalization minutes) upon TNKS inhibition followed by rapid and complete of ubiquitin and b-TrCP with degradasomes. Finally, we used resolution of the complexes after inhibitor washout. Quantitative photobleaching to measure kinetics of exchange between punc- image analysis of individual degradasomes revealed similar expo- tate and diffuse cytoplasmic b-catenin. Similar experiments have nential declines of b-catenin and TNKS fluorescence upon wash- previously been described for AXIN and Dishevelled (46). Our out of the inhibitor. The fluorescence intensities stayed constant photobleaching experiments of G007-LK-induced b-catenin– under control conditions, excluding fluorescence loss due to mCherry puncta revealed 76% recovery of prebleach intensity 1 bleaching. Moreover, resolution of the puncta occurred in parallel within approximately 1 minute after bleaching (T /2: 13.7 sec- with a reduction in both TNKS and AXIN levels. This is most likely onds). Neighboring, nonbleached puncta did not increase in due to a reinitiation of (auto-)poly-ADP-ribosylation activity by size during the time frame of the photobleaching experiments, TNKS resulting in RNF146-mediated ubiquitination and degra- indicating a concomitant high off-rate of degradasome-associated dation of TNKS and AXIN (42–44). The highly reversible nature of b-catenin. Taken together, these results indicate that TNKSi- degradasomes should be taken into account when designing induced degradasomes indeed reflect active sites of b-catenin novel TNKSi, during in vivo experiments and in potential future modification and turnover despite the APC mutation that is clinical trials. present in SW480 cells. We propose that G007-LK-treatment of Live-cell imaging also revealed widespread cytoplasmic move- the SW480 cells restores recruitment of b-TrCP to N-terminal ment of small-sized degradasomes during the initial hours of phosphorylated b-catenin in degradasomes and re-enables b-cate- G007-LK incubation. However, the degradasomes appeared to nin degradation in APC-mutated SW480 cells. Thus, catalytically fuse over time generating larger less mobile complexes. Of note, a inhibited TNKS rescues the functionality of the APC truncation very similar pattern of movement and puncta fusion has previ- in the destruction complex. Interestingly, Faux and colleagues ously been described for Dishevelled/AXIN protein assemblies (49) found that overexpressed monomeric AXINDDIX-RFP can (45, 46). Confocal imaging of fixed cells further showed that both act as a scaffold for destruction complex assembly and b-catenin AXIN2 and b-catenin colocalized with GFP-TNKS1 in puncta phosphorylation in SW480 cells, although AXIN puncta forma- shortly after addition of G007-LK (Supplementary Fig. S8). We tion (overexpressed AXIN-RFP) is required for b-catenin degra- therefore propose that smaller degradasomes are stabilized and dation. Based on these results the authors speculate that puncta accumulate immediately upon TNKS inhibition in SW480 cells formation of AXIN might be required for recruitment, position- and that the puncta observed after 24 hours of treatment represent ing, and/or activation of the E3 ligase complex. Our findings larger assemblies of these complexes. support these models, and experiments are currently underway to Dissociation of the destruction complex due to loss of the determine whether proteasomal degradation of b-catenin takes b-catenin-interacting 20-aa repeat regions (47) and/or the SAMP place in association with puncta or elsewhere in the cytoplasm. repeats of APC that bind AXIN (48) has traditionally been TNKS1/2 have traditionally been regarded as positive regula- regarded as the mechanism of WNT activation in APC-mutated tors of WNT/b-catenin signaling by poly-ADP-ribosylation of colorectal cancer cells. However, Li and colleagues recently pre- AXIN. This leads to a dissociation of the destruction complex sented a contrasting model in which the destruction complex stays and increased levels of nuclear b-catenin. However, several lines of intact in APC-truncated cells, although interaction with the F-box evidence point to a role of TNKSs as structural components of

(Continued.) Statistical testing was based on a mixed effect model and Tukey HSD post hoc test. Incubation with G007-LK together with MG132 for the last hour showed a significant increased level of PCB compared with the other treatments. MG132 treatment alone caused a minor increase in PBC levels when compared with DMSO-treated cells. , P < 0.05; , P < 0.01; , P < 0.0005. B, SW480 GFP-TNKS1 cells were seeded on coverslips and incubated with G007-LK for 24 hours. For the last hour, MG132 was added to one sample. Both samples were immunostained with an antibody against PBC. To obtain images of a high number of cells the coverslips were examined using the ScanR imaging system. A total of 64 images were acquired for each condition, generating around 500 cell profiles per condition. Representative images are shown. Scale bar, 10 mm. Blue, Hoechst. C, quantification of images from B. ScanR analysis software was used to quantify the intensity of both PBC and GFP-TNKS1 within degradasomes. In the graph the intensities of PBC and GFP are shown relative to incubation with G007-LK for 24 hours (values set to 1). Three independent experiments were analyzed (SD) and the data were statistically tested using one-sample t test. PBC intensity in the cells incubated with MG132 together with G007-LK was significantly different than G007-LK alone. , P < 0.01. D, GFP-TNKS1 SW480 cells were seeded on coverslips and incubated with G007-LK for 24 hours, then fixed and immunostained with antibodies against poly-ubiquitin and b-TrCP. Merged image shows GFP-TNKS1 puncta colocalizing with ubiquitin (red) and b-TrCP (white). Scale bar, 2 mm. Blue, Hoechst.

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Figure 6. Induction of degradasomes in different colorectal cancer cell lines. A, confocal sections through COLO320 cells treated with either DMSO (left) or G007-LK (right) for 24 hours and immunostained with antibodies as indicated. Merged images show b-catenin in red and different destruction complex components in green. In contrast to AXIN2 and GSK3, APC does not colocalize with b-catenin in G007-LK-induced degradasomes in COLO320 cells. Destruction complex components show diffuse cytoplasmic staining in control cells. Blue, Hoechst. Scale bar, 2 mm. B, confocal sections through LS174T cells treated with either DMSO (left) or G007-LK (right) for 24 hours and immunostained with antibodies as indicated. All destruction complex components (green) colocalize with b-catenin (red) in G007-LK-induced degradasomes. Blue, Hoechst. Scale bar, 2 mm.

degradasomes: Super-resolution imaging revealed a substructure comparable to the recovery of AXIN (46). Finally, we observed in the G007-LK-induced degradasomes characterized by inter- cytoplasmic protein puncta upon transient overexpression of twined meshworks of TNKS/AXIN and interspersed b-catenin. GFP-TNKS1 in SW480 cells (data not shown). These puncta Electron microscopy further showed an inhomogeneous distri- stained positive for different destruction complex components bution of high electron densities in the protein complexes. More- and were induced in the absence of G007-LK-treatment. over, our FRAP data indicate that TNKS forms a stable component Based on our ﬁndings we suggest that the substructures of degradasomes, as its ﬂuorescence recovery was slow and observed with high resolution imaging in the G007-LK treated

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Figure 7. FRAP reveals different dynamic properties of b-catenin and TNKS in G007-LK-induced degradasomes. A, GFP-TNKS1-expressing SW480 cells were transfected with b-catenin– mCherry and incubated with G007-LK for 24 hours. Individual inhibitor- induced puncta were photobleached and fluorescence recovery of mCherry and GFP within photobleached region (red circle) monitored every 8 seconds. Magnifications show representative fluorescence signals in puncta before photobleaching, immediately after photobleaching and at the end of image acquisition, respectively. B, graph shows average fluorescence recovery values from several individual photobleachings (% of prebleach intensity), corrected for background and normal bleaching due to repetitive acquisition. Red curve: fluorescence recovery of b-catenin-mCherry. Green curve: fluorescence recovery of GFP-TNKS1. Time points are in seconds (00) and 0 indicates the first measured value after photobleaching.

cells represent dense areas of polymerized enzymatically inhib- TNKS in SW480 cells, indicating downstream factors as poten- ited TNKS and known scaffold proteins (AXIN1/2, APC) onto tial candidates for the remaining activity of the pathway. which b-catenin processing can take place. Interestingly, over- Indeed, de la Roche and colleagues recently proposed that high expression of TNKS1 enabled us to visualize cytoplasmic puncta levels of LEF1 and B9L lock a transient burst of b-catenin- resembling TNKSi-induced degradasomes. Consistent with these dependent signaling into a stable state of chronic WNT/b-cate- observations, Callow and colleagues (42) recently showed that nin pathway activity (17). Further studies are required to RNF146 siRNA stabilizes TNKS and induces cytoplasmic protein elucidate if the observed insensitivity to TNKS inhibition is a puncta. Moreover, De Rycker and colleagues (50) previously general phenomenon in colorectal cancer cells or pertain to a demonstrated that TNKS1 can polymerize to assemble large subset of colorectal cancer cell types. The effect of TNKSi in protein complexes. In accordance with these results, we propose other b-catenin-dependent neoplasias and their potential in a novel role of TNKSs as scaffolding proteins in degradasomes. It combination therapy should also be elucidated. will be interesting to investigate in follow-up studies whether In short, our results give novel insight into the molecular enzymatically inactive TNKS can fulfill a structural role and effects of catalytically inhibiting TNKS1/2 in SW480 cells. Our whether depletion or knockout of TNKS will affect degradasome data reveal important structural and kinetic characteristics of formation and/or Wnt/b-catenin signaling. the degradasome and show the potential of TNKSi as a valu- SW480 is one of the most commonly used cell lines in the able tool to study the signal-limiting b-catenin destruction field of TNKSi research. However, despite a substantial reduc- complex. tion in the nonphosphorylated active form of b-catenin upon TNKS inhibition, WNT signaling output is only moderately Disclosure of Potential Conflicts of Interest reduced as measured by luciferase activity assays and WNT The patent for G007-LK is held by Inven2 AS on behalf of the Oslo University target gene mRNA expression levels (15–17, 20). As SW480 Hospital. J. Waaler and S. Krauss are named on the patent. No potential conflicts cells display unusually high levels of b-catenin, the remaining of interest were disclosed by the other authors. fraction after TNKS inhibition might still be sufficient for continued transcription initiation of b-catenin downstream Authors' Contributions genes. Furthermore, we observed some variation in reduction Conception and design: T.E. Thorvaldsen, N.M. Pedersen, E.M. Wenzel, of b-catenin levels between individual cells, indicating that S.W. Schultz, J. Waaler, S. Krauss, H. Stenmark factors such as cell density, cell–cell contacts, and incubation Development of methodology: T.E. Thorvaldsen, N.M. Pedersen, S.W. Schultz conditions might influence the observed effect in individual Acquisition of data (provided animals, acquired and managed patients, experiments. provided facilities, etc.): T.E. Thorvaldsen, N.M. Pedersen, E.M. Wenzel, S.W. Schultz, A. Brech Based on our results and previously published reports, Analysis and interpretation of data (e.g., statistical analysis, biostatistics, b destruction complex activity in -catenin degradation seems computational analysis): T.E. Thorvaldsen, N.M. Pedersen, E.M. Wenzel, to be reestablished when inhibiting the catalytic activity of S.W. Schultz, A. Brech, K. Liestøl

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Writing, review, and/or revision of the manuscript: T.E. Thorvaldsen, Grant Support N.M. Pedersen, E.M. Wenzel, S.W. Schultz, A. Brech, K. Liestøl, J. Waaler, T.E. Thorvaldsen is a PhD student and S.W. Schultz a postdoctoral fellow of S.Krauss,H.Stenmark the South-Eastern Norway Regional Health Authority. E.M. Wenzel is a senior Administrative, technical, or material support (i.e., reporting or organizing research fellow of the South-Eastern Norway Regional Health Authority. data, constructing databases): J. Waaler J. Waaler and S. Krauss are supported by the Research Council of Norway, CRI Study supervision: N.M. Pedersen, H. Stenmark program, and the South Eastern Norway Regional Health Authority, grant 2010031. H. Stenmark has been supported by the Norwegian Cancer Society and by an Advanced Grant from the European Research Council. This work was Acknowledgments partly supported by the Research Council of Norway through its Centres of The authors thank The Core Facilities for Advanced Light Microscopy and Excellence funding scheme, project number 179571. Electron Microscopy at Oslo University Hospital for providing access to The costs of publication of this article were defrayed in part by the payment advertisement relevant microscopes and Kay O. Schink for the customized Fiji script and of page charges. This article must therefore be hereby marked valuable advice. The authors also acknowledge Sascha Beneke for supplying in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. the GFP-TNKS1 plasmid, Ban-Hock Toh for the anti-EEA1 antiserum, Eva Rønning for technical support, and Anne Engen and her co-workers in the Received March 13, 2015; revised May 26, 2015; accepted June 12, 2015; cell lab facility for expert handling of cell cultures. published OnlineFirst June 29, 2015.

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www.aacrjournals.org Mol Cancer Res; 13(11) November 2015 1501

Structure, Dynamics, and Functionality of Tankyrase Inhibitor-Induced Degradasomes

Tor Espen Thorvaldsen, Nina Marie Pedersen, Eva M. Wenzel, et al.

Mol Cancer Res 2015;13:1487-1501. Published OnlineFirst June 29, 2015.

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