An RNAi-based chemical genetic screen identifies three small-molecule inhibitors of the Wnt/wingless signaling pathway

Abstract

Misregulated β-catenin responsive transcription (CRT) has been implicated in the genesis of various malignancies, including colorectal carcinomas, and it is a key therapeutic target in combating various cancers. Despite significant effort, successful clinical implementation of CRT inhibitory therapeutics remains a challenging goal. This is, in part, because of the challenge of identifying inhibitory compounds that specifically modulate the nuclear transcriptional activity of β-catenin while not affecting its cytoskeletal function in stabilizing adherens junctions at the cell membrane. Here, we report an RNAi-based modifier screening strategy for the identification of CRT inhibitors. Our data provide support for the specificity of these inhibitory compounds in antagonizing the transcriptional function of nuclear β-catenin. We show that these inhibitors efficiently block Wnt/β-catenin-induced target genes and phenotypes in various mammalian and cancer cell lines. Importantly, these Wnt inhibitors are specifically cytotoxic to human colon tumor biopsy cultures as well as colon cancer cell lines that exhibit deregulated Wnt signaling.

Fig. 1.

(A) Primary screen. dsRNA-mediated knockdown of Axin results in cytoplasmic stabilization of β-cat, which, on translocation to the nucleus, results in activation of the β-cat responsive dTF12 reporter. (B) dTF12-luciferase activity in response to treatments with a small-molecule library, normalized to plate average, shows that, for all compounds screened, most had little or no effect on Wg signaling. Cut-off for cherry picking lead hits was set at ±0.3 on a logarithmic scale (red dashed line), reflecting either a twofold activation (red oval) or reduction (blue oval). The x axis represents the compounds screened, and the y axis represents the log transformation of the fold change of the dTF12 reporter for individual compounds over that of the plate average. (C) Reporter activity in Cl8 cells transfected with degradation-resistant β-cat is inhibited by candidate inhibitors to a similar extent as in cells transfected with dAxin dsRNA. (D) Western analysis of Dvl2 phosphorylation in Rat2 cells pretreated with iCRTs at the indicated doses and stimulated for 2 h with Wnt3a (Left) or Wnt5a (Right). The upper band, where present, is the phosphorylated form induced by noncanonical Wnt signaling. (E) Candidate inhibitors identified in the primary screen fall into three families: Oxazoles, Thiazoles, and Thiazolidinedione. (E′) Compounds belonging to the Oxazole family (like iCRT3 or C3), which were identified, in the primary screen as strong hits. The majority of them were also tested in secondary specificity and epistasis assays (Fig. S1 B and C). (F–H) Dose-response analysis of the Wnt responsive STF16-luc reporter in the presence of varying concentrations of iCRT3 (F), -5 (G), and -14 (H) in mammalian HEK293 cells.

Fig. 2.

(A) Effect of candidate compounds on the interaction of purified β-cat-His and GST-TCF4. iCRT3, -5, and -14 show a significant inhibitory effect on these interactions compared with nontreated (NT) and DMSO-treated binding reactions. Bottom shows comparable amounts of GST-TCF4 being pulled down. (B) Effect of candidate compounds on β-cat's interaction with endogenous TCF4. HEK293 cells were transfected with S37Aβ-cat and treated overnight with candidate compounds at the indicated concentrations. β-cat immunoprecipitates from whole-cell lysates blotted for endogenous TCF4 show significant reduction in β-cat-TCF4 interaction in the presence of compounds while having negligible effect on β-cat-E-cad interaction. Images are representative of independent experiments and are from different gels. (C and E) Colored squares represent the energy scores (y axis) of the best alternative conformations that were accepted during the flexible Monte Carlo docking simulation for iCRT3 (C) and -5 (E) against each of the 20 pockets on the different crystal structures of β-cat (different colors for different Protein Data Bank crystal structures). Asterisk represents the docked conformation with the best energy score, which for these two compounds, is the lowest energy and is clearly distinguishable (significant energy gap; x axis) from nearby conformations. (D and F) Docked conformations of iCRT3 (D) and -5 (F) in the pocket lined by R469 and K435 on β-cat. (G–I) Traces showing negative derivatives of changes in fluorescence of β-cat-TCF4-SYPRO Orange complex in the presence of indicated concentrations of iCRT3 (red trace in G), -5 (green trace in H), or -14 (orange trace in I). Note the shift in the melting temperature (Tm indicated by arrows; G Inset, H Inset, and I Inset) in presence of iCRTs compared with that in the presence of DMSO (blue trace in G–I).

Fig. 3.

(A) Exposure to Wnt3a causes an evident phenotypic transformation of mouse mammary epithelial cells into long chord-like bundles. (B–F) C57MG cells cultured in the presence of Wnt3a and compounds and/or DMSO stained with DAPI (blue) and FITC-conjugated phalloidin (green) for quantitative high-content image analysis. Candidate compounds inhibit (arrowhead in D–F) the acquisition of the chord-like phenotype seen in DMSO+Wnt3a-treated cells (arrow in C). The algorithm used to quantify this phenotypic transformation identifies intracellular actin fibers (blue overlay in B′–F′) and calculates the anisotropy of the actin fiber alignment within each individual cell (yellow mask outline in B′–F′). (B″–F″) Histogram plot of anisotropy of fiber alignment in cell populations (n ≥ 8,000) treated with individual candidate compounds shows normal distribution in controls; normal peak maxima is highlighted by the square bracket (B″). Treatment with Wnt3a+DMSO results in a skewed distribution of anisotropy (C″), which is rescued by treatment with candidate compounds (D″–F″). (Scale bar: 5 μm.) (G) Quantification of the β-cat target gene, WISP1, in response to treatment with candidate compounds. Error bars show range of variation from mean. (H) MCF7-S37Aβ-cat-HA cells show increased accumulation of Axn2 mRNA compared with control cells, which is inhibited by treatment with candidate compounds. (I) MCF7-S37Aβ-cat-HA cells exhibit invasive potential as assayed by the Boyden chamber invasion assay. Treatment with candidate compounds results in a marked decrease in the number of invading cells.

Fig. 4.

(A) HCT116 cells transfected with siβ-cat show reduced accumulation of Axn2 and CycD1 mRNA, thereby suggesting β-cat–directed transcription of these targets. (B) Inhibition of Axn2, cyc-D1 transcription in response to treatment of HCT116 cells with 50 μM iCRT3 (75 μM for cyc-D1), 50 μM iCRT5 (200 μM for cyc-D1), and 50 μM iCRT14. (A and C) Western blots showing inhibition of β-cat target Cyc-D1 accumulation in HCT116 cells in response to varying concentrations of candidate compounds. iCRTs have no effect on the accumulation or stability of β-cat. Treatment of HCT116 (D–G) and HT29 (H–K) cell lines with 75 μM iCRT3 (E and I), 200 μM iCRT5 (F and J), and 50 μM iCRT14 (G and K) causes a clear cell cycle arrest as shown by flow cytometry. DMSO has no effect on the cell cycle profile of HCT116 (D) and HT29 (H) cells. Red peaks depict cells in G0/G1; yellow depicts G2/M. (L–P and L′–O′) Inhibition of HCT116 proliferation is confirmed by reduction in the number of PH3-positive cells when cultured in the presence of candidate compounds at the indicated concentrations. (Q) Cell cycle arrest caused by compound treatment is also reflected in the reduced number of BrdU-positive cells. (R) Positive or negative SEMs of IC₅₀ values (shown) of iCRT3 compared with that of other drugs show differential sensitivity of colon cancer biopsies from six individual patients to therapeutic agents. Three patient samples show high sensitivity to iCRT3. IC₅₀ values in milligrams per milliliter for iCRT3 in each of the samples tested are depicted above in the graph. 5FU, fluorouracil; CAMP, camptosar; IRES, IRESSA; L-OHP, oxaliplatin; MMC, mitomycin-C; STN, sutent. IC₅₀ values of iCRT3 in individual colon cancer samples are depicted in milligrams per milliliter.