Tankyrase (TNKS) enzymes, due to their poly(ADP-ribose) polymerase activity, have emerged as potential targets in experimental cancer therapy. However, the functional consequences of TNKS inhibition remain incompletely resolved because of the binding promiscuity of TNKS. One of the hallmarks of small-molecule TNKS inhibitors (TNKSi) is the stabilization of AXIN, which plays a pivotal role in the WNT/β-catenin signaling pathway. The present study focused on the known ability of TNKSi to induce cytoplasmic puncta (degradasomes) consisting of components of the signal-limiting WNT/β-catenin destruction complex. Using the colorectal cancer cell line SW480 stably transfected with GFP-TNKS1, it was demonstrated that a TNKS-specific inhibitor (G007-LK) induces highly dynamic and mobile degradasomes that contain phosphorylated β-catenin, ubiquitin, and β-TrCP. Likewise, G007-LK was found to induce similar degradasomes in other colorectal cancer cell lines expressing wild-type or truncated versions of the degradasome component APC. Super-resolution and electron microscopy revealed that the induced degradasomes in SW480 cells are membrane-free structures that consist of a filamentous assembly of high electron densities and discrete subdomains of various destruction complex components. Fluorescence recovery after photobleaching experiments further demonstrated that β-catenin–mCherry was rapidly turned over in the G007-LK-induced degradasomes, whereas GFP-TNKS1 remained stable. In conclusion, TNKS inhibition attenuates WNT/β-catenin signaling by promoting dynamic assemblies of functional active destruction complexes into a TNKS-containing scaffold even in the presence of an APC truncation.

Implications: This study demonstrates that β-catenin is rapidly turned over in highly dynamic assemblies of WNT destruction complexes (degradasomes) upon tankyrase inhibition and provides a direct mechanistic link between degradasome formation and reduced WNT signaling in colorectal cancer cells. Mol Cancer Res; 13(11); 1487–501. ©2015 AACR.

The WNT/β-catenin signaling pathway plays a pivotal role in fundamental biologic processes, including cell proliferation, cell polarity, energy metabolism, and cell fate determination during embryonic development and adult tissue homeostasis (1, 2). Consequently, mutations in this pathway are linked to a broad range of human diseases, including cancer (3). The WNT/β-catenin destruction complex regulates protein turnover of β-catenin, the key mediator of canonical WNT signaling output (4). The structural proteins adenomatous polyposis coli (APC) and axis inhibition protein 1 and 2 (AXIN1/2), and the kinases casein kinase 1α (CK1α) and glycogen synthase kinase 3 (GSK3) are regarded as the core complex components (5). In the WNT-off state, transcriptionally active β-catenin (ABC) levels are kept low by CK1a/GSK3-mediated N-terminal phosphorylation of β-catenin and subsequent degradation by the ubiquitin-proteasome system. Upon WNT activation, ABC escapes N-terminal phosphorylation and proteasomal degradation, translocates to the nucleus, and initiates transcription of WNT/β-catenin-responsive genes by complexing predominantly with the TCF/LEF family of transcription factors (6). The mechanisms by which destruction complex activity is inhibited in the WNT-on state are currently debated (7, 8).

The poly-ADP-ribosyltransferases tankyrase 1 (TNKS1) and tankyrase 2 (TNKS2) modify acceptor proteins by transferring ADP-ribose moieties (poly-ADP-ribosylation) to amino acid side chains. Modified proteins are subsequently poly-ubiquitinated and predominantly turned over by proteasomal degradation (9). TNKS1/2 are involved in a wide range of cellular functions (10) and were recently shown to positively regulate the WNT/β-catenin pathway through poly-ADP-ribosylation of AXIN (11), the rate-limiting factor for destruction complex stability and function (12). Implication of TNKS1/2 as druggable targets in the WNT/β-catenin signaling pathway has generated profound research on developing novel small-molecule inhibitors (9, 13, 14). Inhibition of the catalytic activity of TNKS1/2 reduces WNT/β-catenin signaling in both APC wild-type cells (e.g., in HEK293) and colorectal cancer cells harboring APC truncations (e.g., in SW480; refs. 11, 15, 16). Interestingly, immunofluorescence imaging of colorectal cancer cells has revealed cytoplasmic puncta formation of destruction complex components upon TNKSi treatment (15–17). Based on the presence of phosphorylated β-catenin (phospho-β-catenin [PBC]) in puncta, they have been designated functional degradasomes (18) that promote phosphorylation and subsequent degradation of β-catenin. However, extensive structural and functional studies on these central inhibitor-induced complexes are lacking.

G007-LK is a highly selective TNKSi that attenuates WNT-induced cell growth both in vivo and in vitro (19, 20). In the present study, we used endogenous proteins and stably expressed fluorescent fusion proteins to investigate the molecular effects of G007-LK in APC-truncated SW480 cells. Key experiments were also reproduced in other colorectal cancer cell lines expressing wild-type or truncated versions of APC. We examined the composition and structure of inhibitor-induced degradasomes in SW480 cells by combining confocal, super-resolution, and electron microscopy. Moreover, dynamic properties and functionality of the protein complexes were elucidated by Western blotting, live-cell imaging, and quantitative image analysis. Here, we show that TNKS inhibition by G007-LK induces highly mobile and structurally dynamic degradasomes. Importantly, there is a rapid turnover of β-catenin in the degradasomes as shown by photobleaching experiments. Furthermore, high resolution microscopy enabled us for the first time to reveal structural details of these complexes beyond the resolution limits of confocal microscopy. Our data give novel insight into the mechanisms of TNKS inhibition and attenuation of WNT/β-catenin signaling in colorectal cancer cells and provide a direct mechanistic link between degradasome formation and β-catenin degradation.

Antibodies, plasmids, and chemicals

The following reagents were used: rabbit α-TNKS-1/2 (H-350, specificity validated by colocalization with overexpressed GFP-TNKS1; Santa Cruz Biotechnology); rabbit α-AXIN1 (C95H11, specificity validated by siRNA), rabbit α-AXIN2 (76G6, specificity validated by siRNA), rabbit α-APC, rabbit α-GSK-3β (27C10), rabbit α-PBC (phospho-Ser33/37/Thr41), rabbit α-β-TrCP (specificity validated by colocalization with overexpressed pcDNA3-Flag-β-TrCP; Cell Signaling Technology); mouse α-β-catenin (BD Transduction Laboratories); mouse α-ABC (clone 8E7), mouse α-Ubiquitin (FK1; Millipore); mouse α-β-ACTIN, mouse α-Flag (clone M2; Sigma-Aldrich); rabbit α-p62 (Progen); rabbit α-LC3 (Medical and Biological Laboratories); mouse α-LAMP1 (H4A3; Developmental Studies Hybridoma Bank); rabbit anti-Hrs antiserum (21); human anti-EEA1 antiserum (gift from Ban-Hock Toh, Monash University, Melbourne, Australia; ref. 22); Hoechst (Invitrogen/Dynal); G007-LK (19); MG132 (Chalbiochem); Dimethyl sulphoxide (DMSO) (Sigma Aldrich); GFP-TNKS1 (provided by Sascha Beneke, University of Zurich, Switzerland); pcDNA3-Flag-βTrCP (Addgene, plasmid 10865; ref. 23); β-catenin-mCherry [generated from β-catenin-GFP (24) by standard molecular biology methods]; secondary antibodies for immunofluorescence imaging (Jackson ImmunoResearch Laboratories and Molecular Probes); secondary antibodies for Western blot analysis (IRDye, Li-Cor Biosciences).

Cell-based assays

SW480, COLO320, and LS174T cell lines were purchased from the ATCC. Upon receipt, cells were frozen, and individual aliquots were taken into cell culture, typically for analysis within 15 passages. Cells were grown in RPMI (SW480 and COLO320) or DMEM F12 (LS174T) medium supplemented with 10% (SW480 and COLO320) or 15% (LS174T) FBS and 1% penicillin/streptomycin. Testing for mycoplasma contamination was performed every sixth week. For inhibition of TNKS activity, cells were treated with 0.5 μmol/L G007-LK for 24 hours, unless specified otherwise. DMSO was used as a control. For inhibition of proteasomal activity, cells were treated with 10 μmol/L MG132 for 1 hour, either alone or in combination with G007-LK. To generate a stable SW480 cell line expressing GFP-TNKS1, third-generation lentiviral transduction was used as previously described (25). Detailed cloning procedures can be requested from the authors. Cells were sorted by FACS and frozen. Individual aliquots were grown in cell culture as described for SW480 cells.

Western blot analysis

Cells were rinsed in PBS and lysed in Laemmli lysis buffer [65.8 mmol/L Tris-HCl, pH 6.8, 2.1% SDS, 26.3% (w/v) glycerol, 0.01% bromophenol blue, dithiothreitol (DTT)]. Equal amounts of whole cell lysate were separated by SDS-PAGE (Bio-Rad Laboratories) and blotted with polyvinylidene difluoride membranes (Millipore). Immunodetection was performed with IRDye-conjugated secondary antibodies (LI-COR Biosciences). The Odyssey Imager system (LI-COR Biosciences) was used to scan all blots. Protein bands were quantified using the Odyssey software.

Confocal fluorescence microscopy

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 710/780 microscopes (Carl Zeiss MicroImaging GmbH) using standard filter sets and laser lines and a Plan Apo 63× 1.4NA oil lens. All images were taken at fixed intensity settings below saturation.

Fluorescence recovery after photobleaching (FRAP) experiments

SW480 GFP-TNKS1 cells were seeded in 3.5 cm MatTek glass bottom culture dishes, transfected with β-catenin–mCherry/Lipofectamine 2000 transfection reagent, and treated with G007-LK for 24 hours. A Zeiss LSM 710 confocal microscope was used for photobleaching experiments. Culture dishes were kept in an incubation chamber (5% CO2, 37°C) during the time frame of the experiment. β-catenin–mCherry was bleached with a 561-nm laser line and GFP-TNKS1 with a 405-nm laser line. Fluorescence intensities were measured with the Zen software and data analyzed in the GraphPad Prism software.

Time-lapse live-cell imaging

SW480 GFP-TNKS1 cells were seeded in chambered coverglass with 8 wells (Lab-Tek, Nunc). Unless specified otherwise, RPMI medium with 0.5 μmol/L G007-LK or DMSO (control) was added immediately before imaging of cells with the DeltaVision live-cell imaging system (Applied Precision, GE Healthcare). Washout of inhibitor-medium was performed without removal of the imaging dish from the microscope incubation chamber (5% CO2, 37°C) and imaging was resumed immediately after replacement of medium. Movies were processed and analyzed in the ImageJ/Fiji software (27). A customized Fiji script written in Jython was used to measure the fluorescence intensity of degradasomes over time (28).

ScanR high-throughput microscopy

Cells were grown on coverslips and further processed for antibody staining as described for confocal microscopy samples. Images were automatically taken using the Olympus ScanR system with an UPLSAPO 40×/0.95 objective. All images were taken with the same settings and below pixel saturation. The Olympus ScanR analysis program was used to detect and measure intensity of GFP-TNKS1, AXIN2, and PBC in G007-LK-induced degradasomes. Typically between 400 and 500 cells were analyzed per condition in each experiment.

Statistics

Testing of the different quantifications was done by using mixed effect models (experiments as random factor) for Fig. 4D and for Fig. 5A, in the latter case using Tukey's Honest Significance Difference (HSD) for handling multiple comparisons. The testing was done on log-transformed values and two-sided P values are given in the figure legends. For Fig. 5C, the data were tested with a one-sample t test.

Other materials and methods

See Supplementary Materials and Methods.

G007-LK induces mobile degradasomes and degradation of β-catenin in SW480 colorectal cancer cells

The colorectal cancer cell line SW480 exhibits high cellular levels of β-catenin due to a mutation in the APC gene and is frequently used as a model for WNT-dependent tumors (29). Recent studies have reported AXIN puncta formation and reduced β-catenin levels in SW480 cells upon treatment with different TNKSi (15–17). Consistent with these observations, fluorescence imaging revealed high nuclear β-catenin levels (antibody detecting total β-catenin, hereafter referred to as β-catenin) and low AXIN2 expression in DMSO-treated SW480 cells (Fig. 1A, top). In contrast, G007-LK treatment induced accumulation of β-catenin in cytoplasmic AXIN2-positive puncta, coinciding with reduced nuclear β-catenin. Puncta were distributed throughout the cytoplasm and varied in size with the biggest puncta measuring up to approximately 1 μm in diameter (Fig. 1A, bottom). Western blot analysis confirmed an inverse correlation between G007-LK incubation time and β-catenin levels, with a fraction of the β-catenin pool being resistant to degradation. However, detecting the levels of ABC with an antibody specific for β-catenin dephosphorylated on Ser37 and Thr41 revealed a drastic decline of ABC over time. Indeed, after 24 hours of G007-LK treatment hardly any ABC was present. In contrast, the amount of AXIN2 was substantially increased, accompanied by a moderate increase in AXIN1 levels (Fig. 1B).

Figure 1.

Induction of AXIN puncta and degradation of β-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 β-catenin. Control cells show high nuclear levels of β-catenin and low AXIN2 expression. Treatment with G007-LK increases AXIN2 levels and causes a substantial reduction in the amount of nuclear β-catenin. Magnification of G007-LK-treated cells shows colocalization of AXIN2 (green) with β-catenin (red) in cytoplasmic puncta. Blue, Hoechst. Scale bar, 10 μm. 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 β-catenin levels are progressively reduced upon G007-LK treatment, coincident with increased AXIN1/2 levels. Equal protein loading is documented by staining of β-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.

Figure 1.

Induction of AXIN puncta and degradation of β-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 β-catenin. Control cells show high nuclear levels of β-catenin and low AXIN2 expression. Treatment with G007-LK increases AXIN2 levels and causes a substantial reduction in the amount of nuclear β-catenin. Magnification of G007-LK-treated cells shows colocalization of AXIN2 (green) with β-catenin (red) in cytoplasmic puncta. Blue, Hoechst. Scale bar, 10 μm. 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 β-catenin levels are progressively reduced upon G007-LK treatment, coincident with increased AXIN1/2 levels. Equal protein loading is documented by staining of β-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.

Close modal

To investigate the induction of puncta in more detail we performed live imaging of G007-LK-treated SW480 cells stably expressing GFP-TNKS1 (Fig. 1C), as TNKS1 localizes to inhibitor-induced puncta (Fig. 2A). We chose to take advantage of a stable cell line generated by lentiviral transduction in which the expression of GFP-TNKS1 is under the control of a weak phosphoglycerate kinase 1 promoter in order to ensure even and low expression levels of the fluorescent fusion protein (30). Live-cell imaging revealed a rapid induction of GFP-TNKS1-positive puncta after G007-LK addition (<60 minutes; Fig. 1C, Supplementary Movie S1). Initially, puncta were numerous, small-sized, and barely detectable. These puncta were widespread in the cytoplasm and showed high mobility. However, over a time frame of 7 hours, they appeared to coalesce, generating enlarged structures with perinuclear localization. Of note, puncta mobility gradually declined with increasing size.

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 β-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 μm. B, endosomal and autophagosomal markers (green) do not colocalize with β-catenin (red) in cytoplasmic puncta, as shown in merged images (right). Scale bar, 2 μm. Blue, Hoechst. EEA1, early endosome antigen 1; p62, ubiquitin-binding protein p62.

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 β-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 μm. B, endosomal and autophagosomal markers (green) do not colocalize with β-catenin (red) in cytoplasmic puncta, as shown in merged images (right). Scale bar, 2 μm. Blue, Hoechst. EEA1, early endosome antigen 1; p62, ubiquitin-binding protein p62.

Close modal

To further elucidate the composition of TNKS1 containing puncta, SW480 cells were treated with G007-LK for 24 hours before fixation and immunostaining with antibodies against known destruction complex constituents. Confocal fluorescence microscopy showed AXIN1, AXIN2, GSK3β, TNKS1/2, and APC colocalizing with β-catenin in G007-LK-induced puncta (Fig. 2A), indicating that these structures contain the main components of the WNT destruction complex. Likewise, we observed colocalization of TNKS with endogenous destruction complex components in SW480 cells stably expressing GFP-TNKS1 upon treatment with G007-LK (Supplementary Fig. S1A) but not in SW480 cells treated with DMSO alone (Fig. 2A). Of note, the inhibitor-induced puncta contained both ABC and PBC (Supplementary Fig. S1B). Key experiments were also reproduced with a different chemotype of TNKSi (XAV939; ref. 11). SW480 cells expressing GFP-TNKS1 were treated with XAV939 for 24 hours and stained with antibodies against β-catenin and Axin2. As expected, XAV939-induced puncta contained all three proteins (Supplementary Fig. S1C).

To examine any possible association with endocytic and autophagic pathways, we checked localization of markers of these pathways in G007-LK-treated SW480 cells. However, G007-LK-induced puncta did neither colocalize with markers of early endosomes (EEA1), multivesicular endosomes (HRS), and late endosomes/lysosomes (LAMP1) nor with autophagosomal (LC3/p62) markers that we tested (Fig. 2B and Supplementary Fig. S2). This is consistent with the idea that G007-LK-induced puncta represent proteninaceous, membrane-free structures rather than vesicular compartments. Taken together, we conclude that G007-LK treatment of SW480 cells induces accumulation of destruction complex components in mobile cytoplasmic puncta (hereafter referred to as degradasomes), accompanied by a substantial reduction in ABC.

High resolution imaging reveals novel structural details of degradasomes

In order to investigate the structure of the G007-LK-induced degradasomes at higher resolution, we utilized structured illumination microscopy on the SW480 cells stably expressing GFP-TNKS1 that were treated with G007-LK for 24 hours. Although the degradasomes appeared more or less perfectly round and homogeneous in conventional confocal microscopy, super-resolution microscopy revealed a more complex substructure (Fig. 3A and B): GFP-TNKS, β-catenin and AXIN2 appeared to form intertwined meshworks without showing a particular colocalization between any two of these components.

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 β-catenin and AXIN2. 3D SIM imaging reveals an irregular shape of the induced degradasomes and a nonhomogeneous distribution of GFP-TNKS1 (green), β-catenin (red), and AXIN2 (blue) in subdomains. Displayed is a maximum intensity projection. Scale bar, 1 μm. Individual complexes were enlarged for better visualization (1.2 × 1.2 μm). 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 (+275 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).

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 β-catenin and AXIN2. 3D SIM imaging reveals an irregular shape of the induced degradasomes and a nonhomogeneous distribution of GFP-TNKS1 (green), β-catenin (red), and AXIN2 (blue) in subdomains. Displayed is a maximum intensity projection. Scale bar, 1 μm. Individual complexes were enlarged for better visualization (1.2 × 1.2 μm). 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 (+275 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).

Close modal

To investigate the degradasomes at the ultrastructural level, we treated SW480 cells stably expressing GFP-TNKS1 for 24 hours with G007-LK and examined the induced degradasomes by correlative light and electron microscopy (Fig. 3C–F). Images of degradasomes from confocal microscopy (Fig. 3C) and electron micrographs of the same cell (Fig. 3D) were superimposed, which revealed the fibrillar nature of the degradasome structures and the lack of surrounding membranes (Fig. 3E). Higher magnification revealed that the degradasome consists of a filamentous assembly of high electron densities, possibly interspersed by cytosol (Fig. 3F). In addition, we found membranes in close proximity to the degradasomes. As we had collected 55-nm thick serial sections we were able to obtain a 3D model (Fig. 3G) based on 10 consecutive sections (Supplementary Fig. S3). Following the membranes in 3D it became clear that the membranes identified in each single section (Fig. 3E) are connected with each other and are part of the endoplasmic reticulum (ER). However, there is no indication that these membranes are in direct contact with the degradasome and we can exclude that the degradasome is surrounded by autophagic membranes, which is in line with the lack of autophagic markers (LC3 and p62) as revealed with immunofluorescence (Fig. 2B and Supplementary Fig. S2).

G007-LK-induced degradasomes are rapidly dissolved upon inhibitor-washout

Live microscopy revealed a rapid induction of degradasomes upon TNKSi treatment (Fig. 1C, Supplementary Movie S1). Because the WNT destruction complex is believed to represent a dynamic multiprotein assembly (31), we next tested the stability of the inhibitor-induced puncta in the absence of TNKS inhibition by inhibitor washout experiments. First, SW480 cells expressing GFP-TNKS1 were treated with G007-LK for 4 hours (Fig. 4A, Supplementary Movie S2). Next, cells were washed and the inhibitor-containing medium was replaced with regular RPMI medium. Images were captured every minute for 1 hour following washout with a DeltaVision fluorescence live microscope. Strikingly, the induced degradasomes disappeared gradually in the absence of TNKS inhibition and were hardly detectable after 1 hour (Fig. 4A, Supplementary Movie S2). Moreover, we were able to reinduce complex formation by treating the cells with G007-LK for another 22 hours (Supplementary Fig. S4A).

Figure 4.

G007-LK-induced degradasomes are rapidly dissolved upon inhibitor washout. A, A′: 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. 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 (0′). This underlines the rapid induction of degradasomes upon G007-LK-treatment. A″: 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 μm. B, SW480 GFP-TNKS1 cells coexpressing β-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 μm. 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. In the figure, time point 0′ 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 β-catenin (total). Asterisk: splice variant of TNKS1. β-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 0′ 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 0′ to 90′, in contrast to β-catenin, which did not change significantly after washout.

Figure 4.

G007-LK-induced degradasomes are rapidly dissolved upon inhibitor washout. A, A′: 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. 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 (0′). This underlines the rapid induction of degradasomes upon G007-LK-treatment. A″: 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 μm. B, SW480 GFP-TNKS1 cells coexpressing β-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 μm. 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. In the figure, time point 0′ 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 β-catenin (total). Asterisk: splice variant of TNKS1. β-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 0′ 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 0′ to 90′, in contrast to β-catenin, which did not change significantly after washout.

Close modal

To compare the kinetics of TNKS and β-catenin in degradasomes after washout of the inhibitor, we transfected GFP-TNKS1 expressing cells with β-catenin–mCherry and incubated with G007-LK for 24 hours. Images were then captured with the DeltaVision microscope every minute for 1 hour following either washout of, or continued treatment (control) with, the inhibitor (Fig. 4B, Supplementary Movie S3). We measured the intensity of GFP and mCherry in individual degradasomes (N = 12 [control] + 12 [washout]) at each time point. Quantitative analyses showed that the fluorescence intensity did not change during 1-hour continued treatment with G007-LK. However, after washout of the inhibitor, the GFP and mCherry signals declined in parallel, revealing similar dissociation kinetics for TNKS and β-catenin.

Next, to further characterize the stability of destruction complex components within the degradasomes, GFP-TNKS1 expressing SW480 cells were cultured on coverslips and treated with G007-LK for 24 hours. Individual samples were fixed at 24 hours G007-LK treatment and after 15 and 60 minutes washout with inhibitor-free medium, respectively. All samples were stained for endogenous AXIN2 and β-catenin. Fluorescence microscopy revealed a rapid decline in puncta size and fluorescence intensity for all the three tested complex components (Supplementary Fig. S4B). While β-catenin seemed to redistribute in the cytoplasm and to the nucleus, the GFP-TNKS1 and AXIN2 signal completely disappeared within 1 hour washout.

Our next question was whether the degradasome simply dissolves in the cytoplasm or if the participating proteins are degraded when the TNKSi is removed. To address this, we performed Western blot analysis of SW480 GFP-TNKS1 cells to examine protein levels of central degradasome components (TNKS, AXIN2, and β-catenin) upon washout of G007-LK. Individual samples were lysed and scraped in sample buffer at 24 hours of treatment and up to 90 minutes after washout of the inhibitor. Protein levels were quantified using the Odyssey imaging software and normalized to β-ACTIN. AXIN2 and TNKS protein levels decreased immediately after washout of G007-LK, although there was no significant change in the amount of total β-catenin within the measured time interval (Fig. 4C and D). AXIN is regarded as the rate-limiting factor for destruction complex stability (12) and the rapid degradation of AXIN2 may explain the dissipation of puncta observed by microscopy. We also noticed a substantial reduction in PBC levels after washout of G007-LK, indicating decreased phosphorylation of β-catenin by the destruction complex (Supplementary Fig. S4C). Indeed, ABC levels were completely rescued upon prolonged inhibitor-washout (Supplementary Fig. S4D). Taken together, our results show that TNKSi-induced degradasomes are extremely dynamic and tightly regulated in terms of their assembly and disassembly.

G007-LK-induced degradasomes in SW480 cells represent functional assemblies of destruction complex components targeting β-catenin for proteasomal degradation

In cells with functional destruction complexes, ABC levels are kept low by CK1a/GSK3-mediated N-terminal phosphorylation of β-catenin and subsequent degradation by the ubiquitin-proteasome system. To elucidate the functionality of G007-LK-induced degradasomes in SW480 cells, we decided to investigate phosphorylation of β-catenin within the complexes. Initially, we performed Western blot analysis on SW480 cells incubated with G007-LK for 24 hours and immunoblotted with an antibody specifically detecting PBC (Fig. 5A, lane 2). As β-catenin has been shown to be degraded by proteasomes, the proteasomal inhibitor MG132 was added to G007-LK-containing medium 1 hour before lysis. Inhibition of proteasomal activity showed a substantial increase in PBC protein levels, indicating a high rate of β-catenin turnover in G007-LK-treated cells (Fig. 5A, lane 3). We also observed a minor increase in PBC levels with MG132 treatment alone (Fig. 5A, lane 4), which is in accordance with a modest increase in AXIN levels (data not shown) and previously published observations (32, 33). Protein levels were quantified using the Odyssey imaging software and normalized to β-ACTIN (Fig. 5A, graph).

Figure 5.

Degradasomes target β-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 β-ACTIN (loading control). One representative blot is shown and the graph shows quantification of three independent experiments (±SD). The bars represent PBC related to β-ACTIN. 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 μm. 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 β-TrCP. Merged image shows GFP-TNKS1 puncta colocalizing with ubiquitin (red) and β-TrCP (white). Scale bar, 2 μm. Blue, Hoechst.

Figure 5.

Degradasomes target β-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 β-ACTIN (loading control). One representative blot is shown and the graph shows quantification of three independent experiments (±SD). The bars represent PBC related to β-ACTIN. 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 μm. 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 β-TrCP. Merged image shows GFP-TNKS1 puncta colocalizing with ubiquitin (red) and β-TrCP (white). Scale bar, 2 μm. Blue, Hoechst.

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We next immunostained G007-LK-treated SW480 cells expressing GFP-TNKS1 with the antibody specifically detecting PBC targeted for proteasomal degradation. Confocal microscopy showed that PBC localizes to inhibitor-induced degradasomes (Supplementary Fig. S5A). Based on this, we reasoned that proteasomal inhibition would allow PBC to accumulate in the complexes. The proteasomal inhibitor MG132 was therefore added to G007-LK-containing medium 1 hour before cells were fixed and stained. Indeed, combining MG132 with G007-LK resulted in distinct accumulation of PBC, and an increased colocalization of PBC and GFP-TNKS1 was observed (Supplementary Fig. S5A). The observed effect of proteasomal inhibition was quantified after high-throughput image acquisition using an Olympus ScanR microscope. The intensity of PBC within GFP-TNKS1 puncta was quantified using the ScanR analysis software, and quantitative analysis of fluorescence intensity displayed a 2.5-fold increase in degradasome-associated PBC when including MG132 the last hour of G007-LK-treatment (Fig. 5B and C). In contrast, the fluorescence intensity of GFP-TNKS1 did not change during incubation with G007-LK and MG132 together.

Ubiquitination is a prerequisite for proteasomal degradation of β-catenin (34). To examine whether ubiquitin conjugates colocalize with degradasomes, we immunostained G007-LK-treated GFP-TNKS1-expressing cells with an antibody detecting poly-ubiquitin conjugates. Confocal microscopy revealed ubiquitin-specific staining colocalizing with GFP-TNKS1 (Supplementary Fig. S5B, top).

Based on the presence of ubiquitin, we further stained GFP-TNKS1 expressing SW480 cells with an antibody specifically detecting β-transducing-repeat-containing protein (β-TrCP). β-TrCP is a component of the E3 ubiquitin ligase responsible for ubiquitination of β-catenin in the WNT-off state (34). Indeed, confocal microscopy showed a distinct colocalization of endogenous β-TrCP or overexpressed Flag-β-TrCP with GFP-TNKS1 in the G007-LK-induced degradasomes (Supplementary Fig. S5B, bottom, and S5C). Furthermore, multiple-color fluorescence imaging of inhibitor-treated cells revealed colocalization between GFP-TNKS1, β-TrCP, and Ubiquitin (Fig. 5D). From these data we propose that G007-LK-induced degradasomes in SW480 cells are functional by targeting β-catenin for proteasomal degradation through phosphorylation and ubiquitination.

TNKSi-induced degradasomes in additional colorectal cancer cell lines with wild-type or truncated APC

Interestingly, TNKSi-induced AXIN puncta have also been observed in cells expressing a more severe APC truncation at amino acid 811 that lacks β-catenin and AXIN-binding sites (COLO320 cells; ref. 17). However, these have been termed “stalled degradasomes” due to their inability to promote reduced ABC levels in cells treated with the TNKSi XAV939. We noticed distinct G007-LK-induced AXIN2 puncta in the COLO320 cells, which colocalized with β-catenin, PBC, and GSK3β, but not with APC (Fig. 6A, Supplementary Fig. S6A). In contrast to the findings of de la Roche and colleagues, our Western blot analysis revealed a substantial reduction in ABC levels after G007-LK treatment, accompanied by increased levels of AXIN1/2 (Supplementary Fig. S6B). In line with our findings, Lau and colleagues recently showed strong inhibition of WNT/-β-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 characterize the components and function of degradasomes in COLO320 cells.

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 β-catenin in red and different destruction complex components in green. In contrast to AXIN2 and GSK3, APC does not colocalize with β-catenin in G007-LK-induced degradasomes in COLO320 cells. Destruction complex components show diffuse cytoplasmic staining in control cells. Blue, Hoechst. Scale bar, 2 μm. 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 β-catenin (red) in G007-LK-induced degradasomes. Blue, Hoechst. Scale bar, 2 μm.

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 β-catenin in red and different destruction complex components in green. In contrast to AXIN2 and GSK3, APC does not colocalize with β-catenin in G007-LK-induced degradasomes in COLO320 cells. Destruction complex components show diffuse cytoplasmic staining in control cells. Blue, Hoechst. Scale bar, 2 μm. 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 β-catenin (red) in G007-LK-induced degradasomes. Blue, Hoechst. Scale bar, 2 μm.

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We next investigated the effect of G007-LK in the APC wild-type colorectal cancer cell line LS174T. In these cells, β-catenin is homozygously mutated at Ser45 and therefore cannot be phosphorylated by CK1 and consequently GSK3 (35). Confocal microscopy showed AXIN2 puncta colocalizing with β-catenin, GSK3, and APC in cells treated with G007-LK (Fig. 6B). Moreover, we observed a substantial increase in AXIN1/2 levels by Western blotting (Supplementary Fig. S6C). The levels of ABC did not change upon TNKS inhibition, which was expected due to the β-catenin stabilizing Ser45 mutation in LS174T cells. Our results show that induction of degradasomes upon TNKS inhibition also pertains to APC wild-type cell lines.

β-catenin is constantly turned over in degradasomes

FRAP can be used to investigate protein dynamics and activity in living cells. Fluorescent molecules are irreversibly photobleached in a small area of the cell and subsequent diffusion of surrounding nonbleached fluorescent molecules into the bleached area can be recorded (36). The purpose of including FRAP in our study was to investigate the kinetics of exchange between punctuate (i.e., degradasome-associated) and diffuse cytoplasmic β-catenin during G007-LK-treatment. The SW480 cells stably expressing GFP-TNKS1 were transfected with β-catenin–mCherry. Transfection medium was replaced after 5 hours, followed by 24-hour incubation with G007-LK medium. Transfected cells were investigated with a Zeiss LSM 710 confocal microscope, which revealed a distinct puncta formation. Individual β-catenin–mCherry puncta (N = 32) were photobleached (561 nm laser) to 35% of starting fluorescence and recovered to 76% of prebleach intensity within approximately 1 minute (T½: 13.74 s ± 1.14 SEM). Despite the rapid flow of β-catenin into puncta, fluorescence intensity of unbleached neighboring puncta did not increase during the time frame of these experiments, indicating a concomitant constant outflow of β-catenin (data not shown). GFP-TNKS1 in the same puncta was photobleached (405 nm laser) to 10% of starting fluorescence. However, the GFP signal did not recover within approximately 1 minute after bleaching (Fig. 7A and B, Supplementary Fig. S7). TNKS therefore seems to be considerably more stable than β-catenin in G007-LK-induced puncta, whereas β-catenin is constantly turned over.

Figure 7.

FRAP reveals different dynamic properties of β-catenin and TNKS in G007-LK-induced degradasomes. A, GFP-TNKS1-expressing SW480 cells were transfected with β-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 β-catenin-mCherry. Green curve: fluorescence recovery of GFP-TNKS1. Time points are in seconds (″) and 0 indicates the first measured value after photobleaching.

Figure 7.

FRAP reveals different dynamic properties of β-catenin and TNKS in G007-LK-induced degradasomes. A, GFP-TNKS1-expressing SW480 cells were transfected with β-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 β-catenin-mCherry. Green curve: fluorescence recovery of GFP-TNKS1. Time points are in seconds (″) and 0 indicates the first measured value after photobleaching.

Close modal

Extensive research has generated a detailed understanding of the WNT signaling pathway and its molecular aberrations that underlie cancer development (3). Mutations in the APC gene are found in the majority of colorectal cancer cells (37). Despite this, no targeted therapeutics for APC-mutant colorectal cancer cells have advanced to clinical testing (38). TNKS1/2 were discovered as targets of WNT pathway inhibition in 2009 (11) and several small-molecule TNKSi have been identified in the following years (15, 16, 19, 39–41). TNKSi have been proposed to promote β-catenin degradation through stabilization of AXIN, the concentration limiting factor for destruction complex stability (12). However, studies showing a mechanistic link between AXIN protein stabilization and β-catenin degradation are lacking. Interestingly, immunofluorescence imaging of colorectal cancer cells has recently described puncta of colocalized destruction complex components (degradasomes) upon TNKSi treatment (15–17). Although it is well established that exogenous AXIN puncta are induced upon AXIN overexpression, TNKSi have enabled visualization and characterization of endogenous degradasomes. To elucidate the role of these complexes in reducing aberrant WNT signaling, we focused on APC-mutated SW480 cells treated with the TNKSi G007-LK, but verified our key results in two other cell lines (COLO320 and LS174T) and with a different TNKSi (XAV939). G007-LK is a potent and selective TNKSi, which has shown antitumor efficacy in xenograft and genetically engineered mouse colorectal cancer models (20). In accordance with recent reports, the levels of β-catenin were greatly reduced upon inhibitor treatment, accompanied by increased AXIN levels and induction of degradasomes. High resolution imaging techniques were applied for obtaining novel structural details of the induced degradasomes and revealed a striking substructure with discrete subdomains of various destruction complex components. Moreover, live-cell imaging displayed rapid induction of degradasomes upon TNKSi treatment and their immediate resolution after inhibitor washout. Importantly, quantitative image analyses showed that the G007-LK-induced complexes represent active sites of β-catenin processing, thus providing a direct mechanistic link between degradasome formation and enhanced β-catenin degradation in TNKSi-treated colorectal cancer cells.

Our live-cell imaging approach permitted investigation of TNKSi-induced degradasomes dynamics for the first time. We observed a strikingly rapid induction of degradasomes (<60 minutes) upon TNKS inhibition followed by rapid and complete resolution of the complexes after inhibitor washout. Quantitative image analysis of individual degradasomes revealed similar exponential declines of β-catenin and TNKS fluorescence upon washout of the inhibitor. The fluorescence intensities stayed constant under control conditions, excluding fluorescence loss due to bleaching. Moreover, resolution of the puncta occurred in parallel with a reduction in both TNKS and AXIN levels. This is most likely due to a reinitiation of (auto-)poly-ADP-ribosylation activity by TNKS resulting in RNF146-mediated ubiquitination and degradation of TNKS and AXIN (42–44). The highly reversible nature of degradasomes should be taken into account when designing novel TNKSi, during in vivo experiments and in potential future clinical trials.

Live-cell imaging also revealed widespread cytoplasmic movement of small-sized degradasomes during the initial hours of G007-LK incubation. However, the degradasomes appeared to fuse over time generating larger less mobile complexes. Of note, a very similar pattern of movement and puncta fusion has previously been described for Dishevelled/AXIN protein assemblies (45, 46). Confocal imaging of fixed cells further showed that both AXIN2 and β-catenin colocalized with GFP-TNKS1 in puncta shortly after addition of G007-LK (Supplementary Fig. S8). We therefore propose that smaller degradasomes are stabilized and accumulate immediately upon TNKS inhibition in SW480 cells and that the puncta observed after 24 hours of treatment represent larger assemblies of these complexes.

Dissociation of the destruction complex due to loss of the β-catenin-interacting 20-aa repeat regions (47) and/or the SAMP repeats of APC that bind AXIN (48) has traditionally been regarded as the mechanism of WNT activation in APC-mutated colorectal cancer cells. However, Li and colleagues recently presented a contrasting model in which the destruction complex stays intact in APC-truncated cells, although interaction with the F-box containing protein β-TrCP (substrate recognition subunit for the SCF-TrCP E3 ubiquitin ligases) is disrupted (8). This abrogates ubiquitination of β-catenin and saturates the destruction complex with PBC. Newly synthesized β-catenin thus escapes destruction and initiates transcription of WNT responsive genes. Functionality of TNKSi-induced degradasomes has previously been based on colocalization with PBC and increased PBC protein levels on Western blot analysis (16, 17). However, based on the model of Li and colleagues, these experiments do not prove degradational activity in the complex per se. By implementing high-throughput image acquisition (ScanR microscope), we quantitatively showed that the PBC accumulated in degradasomes when combining TNKSi and proteasomal inhibitors. This was evident by an increased fluorescence intensity and size of the PBC positive puncta. Furthermore, confocal imaging revealed colocalization of ubiquitin and β-TrCP with degradasomes. Finally, we used photobleaching to measure kinetics of exchange between punctate and diffuse cytoplasmic β-catenin. Similar experiments have previously been described for AXIN and Dishevelled (46). Our photobleaching experiments of G007-LK-induced β-catenin–mCherry puncta revealed 76% recovery of prebleach intensity within approximately 1 minute after bleaching (T½: 13.7 seconds). Neighboring, nonbleached puncta did not increase in size during the time frame of the photobleaching experiments, indicating a concomitant high off-rate of degradasome-associated β-catenin. Taken together, these results indicate that TNKSi-induced degradasomes indeed reflect active sites of β-catenin modification and turnover despite the APC mutation that is present in SW480 cells. We propose that G007-LK-treatment of the SW480 cells restores recruitment of β-TrCP to N-terminal phosphorylated β-catenin in degradasomes and re-enables β-catenin degradation in APC-mutated SW480 cells. Thus, catalytically inhibited TNKS rescues the functionality of the APC truncation in the destruction complex. Interestingly, Faux and colleagues (49) found that overexpressed monomeric AXINΔDIX-RFP can act as a scaffold for destruction complex assembly and β-catenin phosphorylation in SW480 cells, although AXIN puncta formation (overexpressed AXIN-RFP) is required for β-catenin degradation. Based on these results the authors speculate that puncta formation of AXIN might be required for recruitment, positioning, and/or activation of the E3 ligase complex. Our findings support these models, and experiments are currently underway to determine whether proteasomal degradation of β-catenin takes place in association with puncta or elsewhere in the cytoplasm.

TNKS1/2 have traditionally been regarded as positive regulators of WNT/β-catenin signaling by poly-ADP-ribosylation of AXIN. This leads to a dissociation of the destruction complex and increased levels of nuclear β-catenin. However, several lines of evidence point to a role of TNKSs as structural components of degradasomes: Super-resolution imaging revealed a substructure in the G007-LK-induced degradasomes characterized by intertwined meshworks of TNKS/AXIN and interspersed β-catenin. Electron microscopy further showed an inhomogeneous distribution of high electron densities in the protein complexes. Moreover, our FRAP data indicate that TNKS forms a stable component of degradasomes, as its fluorescence recovery was slow and comparable to the recovery of AXIN (46). Finally, we observed cytoplasmic protein puncta upon transient overexpression of GFP-TNKS1 in SW480 cells (data not shown). These puncta stained positive for different destruction complex components and were induced in the absence of G007-LK-treatment.

Based on our findings we suggest that the substructures observed with high resolution imaging in the G007-LK treated cells represent dense areas of polymerized enzymatically inhibited TNKS and known scaffold proteins (AXIN1/2, APC) onto which β-catenin processing can take place. Interestingly, overexpression of TNKS1 enabled us to visualize cytoplasmic puncta resembling TNKSi-induced degradasomes. Consistent with these observations, Callow and colleagues (42) recently showed that RNF146 siRNA stabilizes TNKS and induces cytoplasmic protein puncta. Moreover, De Rycker and colleagues (50) previously demonstrated that TNKS1 can polymerize to assemble large protein complexes. In accordance with these results, we propose a novel role of TNKSs as scaffolding proteins in degradasomes. It will be interesting to investigate in follow-up studies whether enzymatically inactive TNKS can fulfill a structural role and whether depletion or knockout of TNKS will affect degradasome formation and/or Wnt/β-catenin signaling.

SW480 is one of the most commonly used cell lines in the field of TNKSi research. However, despite a substantial reduction in the nonphosphorylated active form of β-catenin upon TNKS inhibition, WNT signaling output is only moderately reduced as measured by luciferase activity assays and WNT target gene mRNA expression levels (15–17, 20). As SW480 cells display unusually high levels of β-catenin, the remaining fraction after TNKS inhibition might still be sufficient for continued transcription initiation of β-catenin downstream genes. Furthermore, we observed some variation in reduction of β-catenin levels between individual cells, indicating that factors such as cell density, cell–cell contacts, and incubation conditions might influence the observed effect in individual experiments.

Based on our results and previously published reports, destruction complex activity in β-catenin degradation seems to be reestablished when inhibiting the catalytic activity of TNKS in SW480 cells, indicating downstream factors as potential candidates for the remaining activity of the pathway. Indeed, de la Roche and colleagues recently proposed that high levels of LEF1 and B9L lock a transient burst of β-catenin-dependent signaling into a stable state of chronic WNT/β-catenin pathway activity (17). Further studies are required to elucidate if the observed insensitivity to TNKS inhibition is a general phenomenon in colorectal cancer cells or pertain to a subset of colorectal cancer cell types. The effect of TNKSi in other β-catenin-dependent neoplasias and their potential in combination therapy should also be elucidated.

In short, our results give novel insight into the molecular effects of catalytically inhibiting TNKS1/2 in SW480 cells. Our data reveal important structural and kinetic characteristics of the degradasome and show the potential of TNKSi as a valuable tool to study the signal-limiting β-catenin destruction complex.

The patent for G007-LK is held by Inven2 AS on behalf of the Oslo University Hospital. J. Waaler and S. Krauss are named on the patent. No potential conflicts of interest were disclosed by the other authors.

Conception and design: T.E. Thorvaldsen, N.M. Pedersen, E.M. Wenzel, S.W. Schultz, J. Waaler, S. Krauss, H. Stenmark

Development of methodology: T.E. Thorvaldsen, N.M. Pedersen, S.W. Schultz

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T.E. Thorvaldsen, N.M. Pedersen, E.M. Wenzel, S.W. Schultz, A. Brech

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): T.E. Thorvaldsen, N.M. Pedersen, E.M. Wenzel, S.W. Schultz, A. Brech, K. Liestøl

Writing, review, and/or revision of the manuscript: T.E. Thorvaldsen, N.M. Pedersen, E.M. Wenzel, S.W. Schultz, A. Brech, K. Liestøl, J. Waaler, S. Krauss, H. Stenmark

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Waaler

Study supervision: N.M. Pedersen, H. Stenmark

The authors thank The Core Facilities for Advanced Light Microscopy and Electron Microscopy at Oslo University Hospital for providing access to relevant microscopes and Kay O. Schink for the customized Fiji script and valuable advice. The authors also acknowledge Sascha Beneke for supplying 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 cell lab facility for expert handling of cell cultures.

T.E. Thorvaldsen is a PhD student and S.W. Schultz a postdoctoral fellow of the South-Eastern Norway Regional Health Authority. E.M. Wenzel is a senior research fellow of the South-Eastern Norway Regional Health Authority. J. Waaler and S. Krauss are supported by the Research Council of Norway, CRI 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 partly supported by the Research Council of Norway through its Centres of Excellence funding scheme, project number 179571.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Logan
CY
,
Nusse
R
. 
The Wnt signaling pathway in development and disease
.
Annu Rev Cell Dev Biol
2004
;
20
:
781
810
.
2.
Sethi
JK
,
Vidal–Puig
A
. 
Wnt signalling and the control of cellular metabolism
.
Biochem J
2010
;
427
:
1
17
.
3.
Clevers
H
,
Nusse
R
. 
Wnt/β-Catenin signaling and disease
.
Cell
2012
;
149
:
1192
205
.
4.
Valenta
T
,
Hausmann
G
,
Basler
K
. 
The many faces and functions of β-catenin
.
EMBO J
2012
;
31
:
2714
36
.
5.
Kimelman
D
,
Xu
W
. 
β-catenin destruction complex: insights and questions from a structural perspective
.
Oncogene
2006
;
25
:
7482
91
.
6.
MacDonald
BT
,
Tamai
K
,
He
X
. 
Wnt/β-Catenin signaling: components, mechanisms, and diseases
.
Dev Cell
2009
;
17
:
9
26
.
7.
Metcalfe
C
,
Bienz
M
. 
Inhibition of GSK3 by Wnt signalling—two contrasting models
.
J Cell Sci
2011
;
124
:
3537
44
.
8.
Li
VS
,
Ng
SS
,
Boersema
PJ
,
Low
TY
,
Karthaus
WR
,
Gerlach
JP
, et al
Wnt signaling through inhibition of β-catenin degradation in an intact Axin1 complex
.
Cell
2012
;
149
:
1245
56
.
9.
Haikarainen
T
,
Krauss
S
,
Lehtio
L
. 
Tankyrases: structure, function and therapeutic implications in cancer
.
Curr Pharm Des
2014
;
20
:
6472
88
.
10.
Hsiao
SJ
,
Smith
S
. 
Tankyrase function at telomeres, spindle poles, and beyond
.
Biochimie
2008
;
90
:
83
92
.
11.
Huang
S-MA
,
Mishina
YM
,
Liu
S
,
Cheung
A
,
Stegmeier
F
,
Michaud
GA
, et al
Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling
.
Nature
2009
;
461
:
614
20
.
12.
Lee
E
,
Salic
A
,
Krüger
R
,
Heinrich
R
,
Kirschner
MW
. 
The roles of APC and Axin derived from experimental and theoretical analysis of the Wnt pathway
.
PLoS Biol
2003
;
1
:
e10
.
13.
Lehtiö
L
,
Chi
N-W
,
Krauss
S
. 
Tankyrases as drug targets
.
FEBS J
2013
;
280
:
3576
93
.
14.
Zhan
P
,
Song
YN
,
Itoh
Y
,
Suzuki
T
,
Liu
X
. 
Recent advances in the structure-based rational design of TNKSIs
.
Mol Bio Syst
2014
;
10
:
2783
99
.
15.
Waaler
J
,
Machon
O
,
von Kries
JP
,
Wilson
SR
,
Lundenes
E
,
Wedlich
D
, et al
Novel synthetic antagonists of canonical Wnt signaling inhibit colorectal cancer cell growth
.
Cancer Res
2011
;
71
:
197
205
.
16.
Waaler
J
,
Machon
O
,
Tumova
L
,
Dinh
H
,
Korinek
V
,
Wilson
SR
, et al
A novel tankyrase inhibitor decreases canonical Wnt signaling in colon carcinoma cells and reduces tumor growth in conditional APC mutant mice
.
Cancer Res
2012
;
72
:
2822
32
.
17.
de la Roche
M
,
Ibrahim
AEK
,
Mieszczanek
J
,
Bienz
M
. 
LEF1 and B9L shield β-catenin from inactivation by Axin, desensitizing colorectal cancer cells to Tankyrase inhibitors
.
Cancer Res
2014
;
74
:
1495
505
.
18.
Mendoza-Topaz
C
,
Mieszczanek
J
,
Bienz
M
. 
The Adenomatous polyposis coli tumour suppressor is essential for Axin complex assembly and function and opposes Axin's interaction with Dishevelled
.
Open Biol
2011
;
1
:
110013
.
19.
Voronkov
A
,
Holsworth
DD
,
Waaler
J
,
Wilson
SR
,
Ekblad
B
,
Perdreau-Dahl
H
, et al
Structural basis and SAR for G007-LK, a lead stage 1,2,4-Triazole based specific Tankyrase 1/2 inhibitor
.
J Med Chem
2013
;
56
:
3012
23
.
20.
Lau
T
,
Chan
E
,
Callow
M
,
Waaler
J
,
Boggs
J
,
Blake
RA
, et al
A novel Tankyrase small-molecule inhibitor suppresses APC mutation-driven colorectal tumor growth
.
Cancer Res
2013
;
73
:
3132
44
.
21.
Raiborg
C
,
Grønvold Bache
K
,
Mehlum
A
,
Stang
E
,
Stenmark
H
. 
HRS recruits clathrin to early endosomes
.
EMBO J
2001
;
20
:
5008
21
.
22.
Mu
FT
,
Callaghan
JM
,
Steele-Mortimer
O
,
Stenmark
H
,
Parton
RG
,
Campbell
PL
, et al
EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine “fingers” and contains a calmodulin-binding IQ motif
.
J Biol Chem
1995
;
270
:
13503
11
.
23.
Zhou
P
,
Bogacki
R
,
McReynolds
L
,
Howley
PM
. 
Harnessing the ubiquitination machinery to target the degradation of specific cellular proteins
.
Mol Cell
2000
;
6
:
751
56
.
24.
Johnson
M
,
Sharma
M
,
Jamieson
C
,
Henderson
JM
,
Mok
MTS
,
Bendall
L
, et al
Regulation of β-catenin trafficking to the membrane in living cells
.
Cellular Signalling
2009
;
21
:
339
48
.
25.
Campeau
E
,
Ruhl
VE
,
Rodier
F
,
Smith
CL
,
Rahmberg
BL
,
Fuss
JO
, et al
A versatile viral system for expression and depletion of proteins in mammalian cells
.
PLoS One
2009
;
4
:
e6529
.
26.
Sagona
AP
,
Nezis
IP
,
Pedersen
NM
,
Liestol
K
,
Poulton
J
,
Rusten
TE
, et al
PtdIns(3)P controls cytokinesis through KIF13A-mediated recruitment of FYVE-CENT to the midbody
.
Nat Cell Biol
2010
;
12
:
362
71
.
27.
Schindelin
J
,
Arganda-Carreras
I
,
Frise
E
,
Kaynig
V
,
Longair
M
,
Pietzsch
T
, et al
Fiji: an open-source platform for biological-image analysis
.
Nat Meth
2012
;
9
:
676
82
.
28.
Raiborg
C
,
Wenzel
EM
,
Pedersen
NM
,
Olsvik
H
,
Schink
KO
,
Schultz
SW
, et al
Repeated ER–endosome contacts promote endosome translocation and neurite outgrowth
.
Nature
2015
;
520
:
234
38
.
29.
Morin
PJ
,
Sparks
AB
,
Korinek
V
,
Barker
N
,
Clevers
H
,
Vogelstein
B
, et al
Activation of β-Catenin-Tcf signaling in colon cancer by mutations in β-Catenin or APC
.
Science
1997
;
275
:
1787
90
.
30.
Qin
JY
,
Zhang
L
,
Clift
KL
,
Hulur
I
,
Xiang
AP
,
Ren
B-Z
, et al
Systematic comparison of constitutive promoters and the Doxycycline-inducible promoter
.
PLoS ONE
2010
;
5
:
e10611
.
31.
Stamos
JL
,
Weis
WI
. 
The β-catenin destruction complex
.
Cold Spring Harb Perspect Biol
2013
;
5
:
a007898
.
32.
Sadot
E
,
Conacci-Sorrell
M
,
Zhurinsky
J
,
Shnizer
D
,
Lando
Z
,
Zharhary
D
, et al
Regulation of S33/S37 phosphorylated β-catenin in normal and transformed cells
.
J Cell Sci
2002
;
115
:
2771
80
.
33.
Su
Y
,
Fu
C
,
Ishikawa
S
,
Stella
A
,
Kojima
M
,
Shitoh
K
, et al
APC is essential for targeting phosphorylated β-catenin to the SCFβ-TrCP ubiquitin ligase
.
Mol Cell
2008
;
32
:
652
61
.
34.
Hart
M
,
Concordet
JP
,
Lassot
I
,
Albert
I
,
del los Santos
R
,
Durand
H
, et al
The F-box protein β-TrCP associates with phosphorylated β-catenin and regulates its activity in the cell
.
Curr Biol
1999
;
9
:
207
11
.
35.
Kim
I-J
,
Kang
HC
,
Park
J-H
,
Shin
Y
,
Ku
J-L
,
Lim
S-B
, et al
Development and applications of a β-catenin oligonucleotide microarray: β-catenin mutations are dominantly found in the proximal colon cancers with microsatellite instability
.
Clin Cancer Res
2003
;
9
:
2920
5
.
36.
Reits
EAJ
,
Neefjes
JJ
. 
From fixed to FRAP: measuring protein mobility and activity in living cells
.
Nat Cell Biol
2001
;
3
:
E145
7
.
37.
Cancer Genome Atlas Network
. 
Comprehensive molecular characterization of human colon and rectal cancer
.
Nature
2012
;
487
:
330
7
.
38.
Anastas
JN
,
Moon
RT
. 
WNT signalling pathways as therapeutic targets in cancer
.
Nat Rev Cancer
2013
;
13
:
11
26
.
39.
Chen
B
,
Dodge
ME
,
Tang
W
,
Lu
J
,
Ma
Z
,
Fan
C-W
, et al
Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer
.
Nat Chem Biol
2009
;
5
:
100
7
.
40.
James
RG
,
Davidson
KC
,
Bosch
KA
,
Biechele
TL
,
Robin
NC
,
Taylor
RJ
, et al
WIKI4, a novel inhibitor of Tankyrase and Wnt/β-catenin signaling
.
PLoS One
2012
;
7
:
e50457
.
41.
Shultz
MD
,
Kirby
CA
,
Stams
T
,
Chin
DN
,
Blank
J
,
Charlat
O
, et al
[1,2,4]Triazol-3-ylsulfanylmethyl)-3-phenyl-[1,2,4]oxadiazoles: antagonists of the Wnt pathway that inhibit Tankyrases 1 and 2 via novel adenosine pocket binding
.
J Med Chem
2012
;
55
:
1127
36
.
42.
Callow
MG
,
Tran
H
,
Phu
L
,
Lau
T
,
Lee
J
,
Sandoval
WN
, et al
Ubiquitin ligase RNF146 regulates Tankyrase and Axin to promote Wnt signaling
.
PLoS One
2011
;
6
:
e22595
.
43.
Zhang
Y
,
Liu
S
,
Mickanin
C
,
Feng
Y
,
Charlat
O
,
Michaud
GA
, et al
RNF146 is a poly(ADP-ribose)-directed E3 ligase that regulates axin degradation and Wnt signalling
.
Nat Cell Biol
2011
;
13
:
623
9
.
44.
Yeh
TY
,
Meyer
TN
,
Schwesinger
C
,
Tsun
ZY
,
Lee
RM
,
Chi
NW
. 
Tankyrase recruitment to the lateral membrane in polarized epithelial cells: regulation by cell-cell contact and protein poly(ADP-ribosyl)ation
.
Biochem J
2006
;
399
:
415
25
.
45.
Schwarz-Romond
T
,
Merrifield
C
,
Nichols
BJ
,
Bienz
M
. 
The Wnt signalling effector Dishevelled forms dynamic protein assemblies rather than stable associations with cytoplasmic vesicles
.
J Cell Sci
2005
;
118
:
5269
77
.
46.
Schwarz-Romond
T
,
Metcalfe
C
,
Bienz
M
. 
Dynamic recruitment of axin by Dishevelled protein assemblies
.
J Cell Sci
2007
;
120
:
2402
12
.
47.
Munemitsu
S
,
Albert
I
,
Souza
B
,
Rubinfeld
B
,
Polakis
P
. 
Regulation of intracellular beta-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein
.
Proc Natl Acad Sci U S A
1995
;
92
:
3046
50
.
48.
Behrens
J
,
Jerchow
B-A
,
Würtele
M
,
Grimm
J
,
Asbrand
C
,
Wirtz
R
, et al
Functional interaction of an Axin homolog, Conductin, with β-catenin, APC, and GSK3β
.
Science
1998
;
280
:
596
9
.
49.
Faux
MC
,
Coates
JL
,
Catimel
B
,
Cody
S
,
Clayton
AHA
,
Layton
MJ
, et al
Recruitment of adenomatous polyposis coli and β-catenin to axin-puncta
.
Oncogene
2008
;
27
:
5808
20
.
50.
De Rycker
M
,
Price
CM
. 
Tankyrase polymerization is controlled by its sterile alpha motif and poly(ADP-ribose) polymerase domains
.
Mol Cell Biol
2004
;
24
:
9802
12
.

Supplementary data