WIKI4, a novel inhibitor of tankyrase and Wnt/ß-catenin signaling - PubMed (original) (raw)

WIKI4, a novel inhibitor of tankyrase and Wnt/ß-catenin signaling

Richard G James et al. PLoS One. 2012.

Abstract

The Wnt/ß-catenin signaling pathway controls important cellular events during development and often contributes to disease when dysregulated. Using high throughput screening we have identified a new small molecule inhibitor of Wnt/ß-catenin signaling, WIKI4. WIKI4 inhibits expression of ß-catenin target genes and cellular responses to Wnt/ß-catenin signaling in cancer cell lines as well as in human embryonic stem cells. Furthermore, we demonstrate that WIKI4 mediates its effects on Wnt/ß-catenin signaling by inhibiting the enzymatic activity of TNKS2, a regulator of AXIN ubiquitylation and degradation. While TNKS has previously been shown to be the target of small molecule inhibitors of Wnt/ß-catenin signaling, WIKI4 is structurally distinct from previously identified TNKS inhibitors.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have read the journal’s policy and have the following conflicts: RTM is a co-founder of, and consultant with, FATE THERAPEUTICS, San Diego.

Figures

Figure 1

Figure 1. WIKI4 is identified as a novel small molecule inhibitor of the Wnt/ß-catenin pathway.

(A) Scatter plot of a small molecule screen in human A375 melanoma cells stably expressing the ß-catenin Activated Reporter (BAR) driving firefly luciferase with each dot representing a single compound. The red dots represent compounds that exhibited decreased luciferase signal (> two standard deviations below the sample mean), and unchanged cell viability as measured by resazurin. (B) A heat map showing the effects of five Wnt/ß-catenin inhibitors on reporters for the Wnt/ß-catenin, Nuclear Factor Kappa B (NF-kB), Retinoic Acid (RA), and Transforming Growth Factor ß (TGFB) pathways. WIKI4 (arrow) is the only compound that specifically inhibits Wnt/ß-catenin signaling. (C) Chemical structure of WIKI4 (left) and XAV-939 (right). (D) Dose response curves showing that WIKI4 inhibits ß-catenin reporter activity in DLD1 colorectal carcinoma cells and Wnt-stimulated A375 melanoma cells. (E) Inhibition of the expression of the ß-catenin target genes AXIN2 and TNFRSF19 by WIKI4 as assessed by quantitative PCR. DLD cells were transfected with CTNNB1 siRNA as a control 72 hours prior to harvesting for RNA; cells were treated with compounds or DMSO for 16 hours prior to harvesting. The experiments in (D) and (E) are representative of three independent experiments and the error bars represent standard deviation from four technical replicates.

Figure 2

Figure 2. WIKI4 inhibits the functional outcomes of Wnt/ß-catenin signaling.

(A) WIKI4 inhibits colony formation of DLD1 colorectal cancer cells. DLD1 cells were plated individually in 0.5% serum containing medium, and treated with the indicated concentrations of WIKI4 and XAV-939. This experiment is representative of three independent experiments and the error bars represent standard deviation of three technical replicates. (B-F) WIKI4 prevents Wnt3A-dependent differentiation of H1 human embryonic stem cells (hESCs). (B) Culturing hESCs for six days with Wnt3A causes marked morphological changes that are rescued by treatment with WIKI4. Scalebar = 500 µm. (C) Treatment with WIKI4 prevents the decrease in co-expression of markers of undifferentiated hESCs following Wnt3A stimulus. hESCs were stimulated with the indicated treatments and expression of GCTM2 and CD9 was assessed by flow cytometry following six days of treatment. (D-F) The effect of WIKI4 treatment on the expression of genes that are altered during Wnt3A-dependent differentiation of hESCs was assessed by qPCR. hESCs were treated for the indicated conditions for six days, and then analyzed by qPCR for markers of undifferentiated stem cells (NANOG, POU5F1) (D), endoderm (SOX17, GATA6) (E), and mesoderm (T, KDR) (F). The data was normalized to 100,000 copies of GAPDH and plotted as a ratio to the untreated hESCs (cultured in KSR media). The data in the experiments presented in B-F are representative of three independent experiments and the error represents standard deviation of technical replicates. In B-F, LCM = control L cell CM, WNT3A = Wnt3a CM; both 50% (vol/vol) in KSR medium.

Figure 3

Figure 3. WIKI4 increases the steady-state abundance of the Wnt/ß-catenin inhibitory protein, AXIN1.

(A) WIKI4 prevents degradation of AXIN1 following stimulation with Wnt3A. A375 melanoma cells were stimulated with 10% (vol/vol) Wnt3A CM for the indicated time periods with or without WIKI4 treatment, lysed and analyzed by western blot using the indicated antibodies. (B) WIKI4 increases the steady-state abundance of AXIN1 and AXIN2 protein. DLD1 colorectal carcinoma cells were incubated with DMSO, WIKI4 or XAV-939 for the indicated times, lysed and analyzed by western blot. (C) WIKI4 does not significantly affect the steady-state RNA abundance of AXIN1. DLD1 colorectal carcinoma cells were incubated with WIK4 for the indicated times, and processed for qPCR to assess changes in the steady-state abundance of AXIN1 transcript. This data is representative of two independent experiments and the error bars represent standard deviation. (D) WIKI4-dependent increases in AXIN1 protein abundance can be maintained by treatment with a proteasome inhibitor. DLD1 colorectal carcinoma cells were treated overnight with WIKI4, and after washing were then incubated for two hours with DMSO (D), WIKI4 (W), or the proteasome inhibitor MG132 (M). The cells were lysed and analyzed by western blotting for the indicated antibodies.

Figure 4

Figure 4. WIKI4 prevents ubiquitylation of AXIN and inhibits the enzymatic activity of TNKS2.

(A) Schematic showing a model of how AXIN proteins are sequentially ADP-Ribosylated and then poly-ubiquitylated prior to their degradation by the proteasome. (B) WIKI4 inhibits ubiquitylation of AXIN2 in SW480 colorectal carcinoma cells. SW480 cells were treated overnight with DMSO (D), 2.5 µM WIKI4 (W) or 2.5 µM XAV-939 (X). Following a brief wash, the cells were then incubated for two hours with DMSO (D), 10 µM MG132 (M) or MG132 and one of the Wnt/ß-catenin pathway inhibitors. Lysates and AXIN2 immunoprecipitates from this experiment were processed for western blotting with the indicated antibodies. (C) WIKI4 inhibits the enzymatic activity of TNKS2. Recombinant GST-TNKS2 was bound to 96-well plates coated with glutathione. Auto-ADP-ribosylation assays were carried out using biotinylated substrate in the context of the indicated treatments. The amount of TNKS2 auto-ribosylation was quantified by performing chemiluminescent detection of the reaction between streptavidin conjugated to horseradish peroxidase and biotinylated substrate. U0126 was used as a negative control.

Similar articles

Cited by

References

    1. MacDonald BT, Tamai K, He X (2009) Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 17: 9–26. - PMC - PubMed
    1. Tanaka SS, Kojima Y, Yamaguchi YL, Nishinakamura R, Tam PP (2011) Impact of WNT signaling on tissue lineage differentiation in the early mouse embryo. Dev Growth Differ 53: 843–856. - PubMed
    1. Clevers H (2006) Wnt/beta-catenin signaling in development and disease. Cell 127: 469–480. - PubMed
    1. Maretto S, Cordenonsi M, Dupont S, Braghetta P, Broccoli V, et al. (2003) Mapping Wnt/beta-catenin signaling during mouse development and in colorectal tumors. Proc Natl Acad Sci U S A 100: 3299–3304. - PMC - PubMed
    1. Shimizu N, Kawakami K, Ishitani T (2012) Visualization and exploration of Tcf/Lef function using a highly responsive Wnt/beta-catenin signaling-reporter transgenic zebrafish. Dev Biol. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources