Small-molecule synergist of the Wnt/beta-catenin signaling pathway - PubMed (original) (raw)

. 2007 May 1;104(18):7444-8.

doi: 10.1073/pnas.0702136104. Epub 2007 Apr 25.

Michael B Major, Shinichi Takanashi, Nathan D Camp, Naoyuki Nishiya, Eric C Peters, Mark H Ginsberg, Xiaoying Jian, Paul A Randazzo, Peter G Schultz, Randall T Moon, Sheng Ding

Affiliations

Small-molecule synergist of the Wnt/beta-catenin signaling pathway

Qisheng Zhang et al. Proc Natl Acad Sci U S A. 2007.

Erratum in

Abstract

The Wnt/beta-catenin signaling pathway regulates cell fate and behavior during embryogenesis, adult tissue homeostasis, and regeneration. When inappropriately activated, the pathway has been linked to colorectal cancer and melanoma, and when attenuated it may contribute to Alzheimer's disease and osteoporosis. Small molecules that modulate Wnt signaling will likely provide new insights into the regulation of this key developmental pathway and ultimately provide pharmacological agents to control Wnt signaling in vivo. To this end, we screened a library of 100,000 small molecules for activity in a cell-based assay of Wnt/beta-catenin signaling and discovered a purine derivative, QS11, that synergizes with Wnt-3a ligand in the activation of Wnt/beta-catenin signal transduction. Through affinity chromatography and subsequent functional assays, we showed that QS11 binds and inhibits the GTPase activating protein of ADP-ribosylation factor 1 (ARFGAP1), suggesting that QS11 modulates Wnt/beta-catenin signaling through an effect on protein trafficking. Consistent with its function as an ARFGAP inhibitor, QS11 inhibits migration of ARFGAP overexpressing breast cancer cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

QS11 is a small-molecule Wnt synergist in cell culture and Xenopus development. (A) Chemical structures of the purine derivative, QS11, and its negative control, QS11-NC. (B) Dose-dependent effects of QS11 on HEK293 cells transfected with Super(8X)TOPFlash reporter (with Wnt-3a CM, blue line; without Wnt-3a CM, red line) or SuperFOPFlash reporter (with Wnt-3a CM, yellow line). The cells were treated with QS11 at the indicated concentrations with or without Wnt-3a stimulation 24 h after transfection. Luciferase activity was measured 36 h after compound treatment. All data are normalized against renilla luciferase. Error bars are SD. (C) Synergistic effect of QS11 with XWnt-8 RNA on axis duplication in Xenopus. Xenopus embryos were treated with QS11 (10 μM, 10 nL), XWnt-8 RNA (0.5 pg), and QS11 (10 μM, 10 nL) plus XWnt-8 RNA (0.5 pg) for 24 h. (Upper) Representative pictures of Xenopus with different degrees of axis duplication. (Lower) Quantification of embryonic axis duplication on treatment with QS11 (10 μM, 10 nL) and/or XWnt-8 RNA (0.5 pg).

Fig. 2.

Fig. 2.

Affinity chromatography identified ARFGAP1 as the cellular target of QS11. (A) Chemical structures of affinity resins with QS11 (positive resin, M-1) or QS11-NC (negative resin, M-2) immobilized for target identification. (B) Pull-down experiments using the immobilized reagents M-1 [lane M-1, without soluble QS11; lane M-1 + QS11 (50 μM), with soluble QS11 at 50 μM] and M-2 (lane M-2). HEK293 cell lysates were incubated with the affinity matrices at 4°C for 1 h. Bound proteins were eluted, resolved on a 4–20% Tris-glycine gel, and visualized with silver staining. The band that contains ARFGAP1 is indicated by the arrow. (C) Western blot of ARFGAP1 resin-bound protein. Proteins that were resolved on a 4–20% Tris-glycine gel as in B were transferred to a nitrocellulose membrane and analyzed with an antibody against ARFGAP1. (D) Overexpression of ARFGAP1 cDNA blocks the synergistic effect of QS11 with Wnt-3a CM. HEK293 cells were transfected with Super(8X)TOPFlash reporter, pTK-RL plasmid, and ARFGAP1 cDNA (red line) or ssDNA (blue line) by using Fugene6. Cells were treated with QS11 at the indicated concentrations and Wnt-3a CM (1:1 vol/vol ratio to the growth medium) 24 h after transfection. Luciferase activities were measured 36 h after treatment with QS11 and Wnt-3a CM. The activation fold was normalized against renilla luciferase. Error bars are SD.

Fig. 3.

Fig. 3.

QS11 synergizes with Wnt-3a in activating Wnt signaling through ARF activation. (A) QS11 increased cellular ARF-GTP levels. NIH 3T3 cells were treated with QS11, DMSO, or QS11-NC at the indicated concentrations for 36 h. The cell lysates were analyzed with an antibody against ARF1 or ARF6 before (total ARF1 or ARF6) and after (ARF1-GTP or ARF6-GTP) incubating with a GST-fusion protein GGA3VHS-GAT. (B) HEK293 cells were transfected with the Super(8X)TOPFlash reporter, pTK-RL plasmid, and cDNA of arfaptin 1 (red line) or ssDNA (blue line) using Fugene6. The cells were treated with QS11 at the indicated concentrations and Wnt-3a CM (1:1 vol/vol ratio to the growth medium) 24 h after transfection. Luciferase activities were measured 36 h after QS11 treatment. The activation fold was normalized against renilla luciferase. Error bars are SD. (C) The effect of QS11 on nuclear β-catenin. HEK293 cells were treated with QS11 at the indicated concentrations and Wnt-3a CM (1:1 vol/vol ratio to the growth medium) for 24 h. Nuclear β-catenin was analyzed with an antibody against β-catenin. γ-tubulin was used as the loading control.

Fig. 4.

Fig. 4.

QS11 inhibits migration of AMAP1 overexpressing MDA-MB-231 cells. Dose-dependent inhibition of the migration of AMAP1 overexpressing MDA-MB-231 cells by QS11 treatment in a transwell assay. Migration was measured with a modified Boyden chamber containing Transwell filters (Coastar) coated on the underside with 5 μg/cm2 Matrigel. Migrated cells were counted under a microscope.

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