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A Novel Tankyrase Inhibitor Decreases Canonical Wnt Signaling in Colon Carcinoma Cells and Reduces Tumor Growth in Conditional APC Mutant Mice

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A Novel Tankyrase Inhibitor Decreases Canonical Wnt Signaling in Colon Carcinoma Cells and Reduces Tumor Growth in Conditional APC Mutant Mice Published OnlineFirst March 22, 2012; DOI: 10.1158/0008-5472.CAN-11-3336 Cancer Therapeutics, Targets, and Chemical Biology Research A Novel Tankyrase Inhibitor Decreases Canonical Wnt Signaling in Colon Carcinoma Cells and Reduces Tumor Growth in Conditional APC Mutant Mice Jo Waaler1, Ondrej Machon1,6, Lucie Tumova6, Huyen Dinh1, Vladimir Korinek6, Steven Ray Wilson2, Jan Erik Paulsen3, Nina Marie Pedersen4, Tor J. Eide5, Olga Machonova1,6, Dietmar Gradl7, Andrey Voronkov1, Jens Peter von Kries8, and Stefan Krauss1 Abstract Increased nuclear accumulation of b-catenin, a mediator of canonical Wnt signaling, is found in numerous tumors and is frequently associated with tumor progression and metastasis. Inhibition of Wnt/b-catenin signaling therefore is an attractive strategy for anticancer drugs. In this study, we have identified a novel small molecule inhibitor of the b-catenin signaling pathway, JW55, that functions via inhibition of the PARP domain of tankyrase 1 and tankyrase 2 (TNKS1/2), regulators of the b-catenin destruction complex. Inhibition of TNKS1/2 poly(ADP-ribosyl)ation activity by JW55 led to stabilization of AXIN2, a member of the b-catenin destruction complex, followed by increased degradation of b-catenin. In a dose-dependent manner, JW55 inhibited canonical Wnt signaling in colon carcinoma cells that contained mutations in either the APC (adenomatous polyposis coli) locus or in an allele of b-catenin. In addition, JW55 reduced XWnt8-induced axis duplication in Xenopus embryos and tamoxifen-induced polyposis formation in conditional APC mutant mice. Together, our findings provide a novel chemotype for targeting canonical Wnt/b-catenin signaling through inhibiting the PARP domain of TNKS1/2. Cancer Res; 1–11. Ó2012 AACR. Introduction tion complex consisting of adenomatous polyposis coli (APC), b a The Wnt/b-catenin signaling pathway is a key regulator in AXIN2, GSK3 , and CK1 (4, 5). Tankyrase 1 and 2 (TNKS1/2) b numerous cellular processes including stem cell maintenance, regulate the stability of the -catenin destruction complex fate decision, and cell-cycle control (1, 2). The main denom- through AXIN2 by poly(ADP-ribosyl)ation (6). Although inator of canonical Wnt signaling, b-catenin, has several TNKS1/2 modulates the destruction complex, this complex cellular functions. At the cell membrane, it is associated with interferes with TNKS. Evidence suggests that TNKS is phos- – b E-cadherin and participates in the formation of the adherens phorylated in a cell-cycle dependent manner by GSK3 (7). junctions (3). In the cytoplasm, b-catenin can form complexes TNKS is furthermore controlled by the ubiquitin E3 ligase with a multitude of proteins, including the b-catenin destruc- RNF146 that destabilizes TNKS1/2, AXIN2, and itself by ubi- quitinylation (8, 9). In the standard model, the destruction complex regulates the sequential phosphorylation of b-catenin Authors' Affiliations: 1Oslo University Hospital, SFI-CAST Biomedical by the kinases CK1a at S45 and by GSK3b at S33, S37, and T41. Innovation Center, Unit for Cell Signaling, Forskningsparken, Gaustadal- b leen and Center for Molecular Biology and Neuroscience; 2Department of This leads to polyubiquitination by -TrCP and subsequent Chemistry, University of Oslo; 3Norwegian School of Veterinary Science, degradation of b-catenin by the proteasome (10, 11). Active Department of Food Safety and Infection Biology; 4Institute for Cancer Wnt signaling inhibits the function of the destruction complex, Research, Department of Biochemistry, The Norwegian Radium Hospital, b Stem Cell Innovation Centre, 5Department of Pathology, Rikshospitalet, and -catenin is not phosphorylated at the N-terminus and Oslo University Hospital, Oslo, Norway; 6Institute of Molecular Genetics, degraded, but enters the nucleus. Nuclear uptake of b-catenin 7 Czech Academy of Sciences, Prague, Czech Republic; KIT (Karlsruhe may be further enhanced by the context-dependent C-terminal Institute of Technology) Zoological Institute, Cell and Developmental Biol- ogy, Karlsruhe; and 8Leibniz-Institut Fur€ Molekulare Pharmakologie, FMP, phosphorylation of b-catenin at S675, leading to increased Berlin, Germany nuclear translocation (12). In addition to canonical Wnt sig- Note: Supplementary data for this article are available at Cancer Research naling, a variety of alternative cellular mechanisms may trigger Online (http://cancerres.aacrjournals.org/). altered nuclear accumulation of b-catenin. These mechanisms Corresponding Authors: Jo Waaler, Oslo University Hospital, SFI-CAST include the Hif-1a signaling pathway, downregulation of E- Biomedical Innovation Center, Unit for Cell Signaling, Forskningsparken, cadherin or C-terminal phosphorylation of b-catenin through Gaustadalleen, 21, 0349 Oslo, Norway and Center for Molecular Biology – and Neuroscience, P.O. Box 1105, Oslo N-0317, Norway. Phone: 47-9514- HGF-activated cMet (13 15). Furthermore, PKA-involving 8669; Fax: 47-2295-8150; E-mail: [email protected]; and Stefan mechanisms can lead to C-terminal phosphorylation of b-cate- Krauss, E-mail: [email protected] nin and enhance its nuclear presence (12). Recently it was doi: 10.1158/0008-5472.CAN-11-3336 discovered that the primary cilium diverts Jouberin from the Ó2012 American Association for Cancer Research. nucleus and limits b-catenin nuclear entry (16). www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst March 22, 2012; DOI: 10.1158/0008-5472.CAN-11-3336 Waaler et al. In the nucleus, b-catenin exerts a number of functions, such 10 mmol/L JW55 for RKO cells and 0.1% DMSO or 10, 5, or as binding to the transcription factor TCF/LEF by replacing 1 mmol/L JW55 for SW480 cells. All samples consisted of a Groucho and activation of downstream transcription of genes, minimum of 6 replicates. The plate was incubated in an Incu- including c-Myc, Cyclin D1, and AXIN2 (2). Nuclear b-catenin Cyte (Essen BioScience) inside a cell culture incubator. Images levels are not only determined by the rate of b-catenin import were captured every second hour to monitor proliferation. to the nucleus but also by "nuclear trapping" of b-catenin through factors such as TCF/LEF (17). Colony assay In general, mutations in genes encoding central components A total of 400 SW480, RKO, or HeLa cells were seeded in 6-cm of the Wnt/b-catenin pathway and the destruction complex plates and 1% FBS. The day after, the medium was exchanged lead to the accumulation of nuclear b-catenin and contribute to 0.05% DMSO or 5 mmol/L JW55 for RKO and HeLa cells, and to tumor initiation and progression (18, 19). Wnt activating 0.1% DMSO or 5, 1, or 0.1 mmol/L JW55 for SW480 cells mutations are found in a broad range of solid tumors including (minimum 3 replicates). The cells were grown until colonies colon cancer, gastric cancer, hepatocellular carcinoma, breast appeared in the control plates and medium was changed twice cancer, medulloblastoma, melanoma, non–small cell lung can- a week. The plates were stained and fixed with 0.2% methylene cer, pancreas adenocarcinoma, and prostate cancer (20). It has blue in methanol and washed with PBS. The colonies were been well established that the majority of intestinal neoplasia counted manually with a colony counter (Scienceware). Rep- with deregulated Wnt signaling harbor truncating mutations in resentative pictures of colonies were captured with a Leica MZ both alleles of the APC tumor suppressor gene (21). Apo Stereomicroscope connected to a Leica DC200 camera Considerable efforts have been made to identify drugs and Adobe Photoshop. that inhibit Wnt/b-catenin signaling (18, 22–25). Published inhibitory drugs that interact with biotargets directly asso- Tumor initiation and treatment in ApcCKO/CKO/Lgr5- ciated with canonical Wnt signaling include WAY-316606 CreERT2 mice for SFRP (26), niclosamide for frizzled (27), NCS668036 Seven 12-week old female ApcCKO/CKO/Lgr5-CreERT2 mice for Dsh (28), pyrvinium for CK1 (24), XAV939 and IWR-1 were injected intraperitonally with 25 mg/kg of tamoxifen for TNKS1/2 (4, 22), 2,4-diamino-quinazoline, quercetin, (Sigma) diluted in an ethanol and corn oil (ratio 1:4). The mice b PKF115-584 and ICG-001 for -catenin and its binding to were randomized into 2 groups and treated with either JW55 – TCF or CREB (29 32). (100 mg/kg) or vehicle (DMSO). Daily per oral applications TNKS1/2, in particular, is a promising biotarget for phar- started the day after and continued for 3 weeks. The mouse maceutical reagents (4, 6). TNKS1/2 is not only involved in body weight was measured twice a week. The mice were controlling canonical Wnt signaling but has been associated sacrificed and the intestines were dissected, washed in PBS, with further cellular processes through either protein poly and fixed in formaldehyde [10% solution (v/v) in PBS]. The (ADP-ribosyl)ation (PARsylation) or by protein complex for- small intestines were stained using 1% methylene blue pre- mation. These are (i) telomere maintenance by interacting pared in 10% paraformalaldehyde (PFA)/PBS solution. Small with TRF1 (33), (ii) spindle formation and stabilization by ileum Swiss-rolls were embedded in paraffin sectioned and binding to NuMA (33), and (iii) glucose metabolism by regu- stained with hematoxylin and eosin (H&E). Fixed colons were lating GLUT4 transport through IRAP (33). embedded in paraffin, sectioned and stained with an anti– fi In this work, we have identi ed a novel TNKS inhibitor, b-catenin antibody (610153; BD Transduction Laboratories; JW55, which inhibits the PARP domain of TNKS1/2, leading to see Supplementary Materials and Methods for further details). the stabilization of AXIN2 followed by increased degradation of The number and size of the intestinal lesions were quantified b fi -catenin. JW55 ef ciently decreases canonical Wnt signaling by the Ellipse program (ViDiTo). in colon carcinoma cell lines, in a Xenopus axis duplication For other materials and methods, please see previous assay, and in tamoxifen-induced tumors in ApcCKO/CKOLgr5- þ descriptions (34) and Supplementary Materials and Methods.
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