A small molecule inhibitor of beta-catenin/CREB-binding protein transcription [corrected]

Abstract

Inherited and somatic mutations in the adenomatous polyposis coli occur in most colon cancers, leading to activation of beta-catenin-responsive genes. To identify small molecule antagonists of this pathway, we challenged transformed colorectal cells with a secondary structure-templated chemical library, looking for compounds that inhibit a beta-catenin-responsive reporter. We identified ICG-001, a small molecule that down-regulates beta-catenin/T cell factor signaling by specifically binding to cyclic AMP response element-binding protein. ICG-001 selectively induces apoptosis in transformed cells but not in normal colon cells, reduces in vitro growth of colon carcinoma cells, and is efficacious in the Min mouse and nude mouse xenograft models of colon cancer.

Fig. 1.

ICG-001 binds CBP and inhibits β-catenin/TCF-mediated transcription. (A) Chemical structures of ICG-001 and ICG-002. (B and C) ICG-001 selectively inhibits the β-catenin/TCF reporter construct TOPFLASH but not FOPFLASH. (D) CBP is the molecular target of ICG-001. Nuclear extracts of SW480 cells were incubated with streptavidin-agarose beads coated with ICG-002. Bound proteins were eluted and immunoblotted (4% gel) with anti-CBP antibody (Upper) or silver-stained (Lower).

Fig. 2.

ICG-001 binds CBP and selectively disrupts the β-catenin/CBP interaction. (A) SW480 cells were transfected with full-length β-catenin (2.2 μg) or full-length CBP (1.1 μg). Nuclear lysates (50 μg) were treated with 20 μM ¹⁴C-labeled-ICG-001 alone (¹⁴C-ICG-001) or with 100 μM cold ICG-001. Unbound ¹⁴C-labeled-ICG-001 was removed, and ¹⁴C-labeled ICG-001 incorporation was measured. (B) ICG-001 specifically disrupts the CBP/β-catenin interaction without disrupting the p300/β-catenin. CBP (amino acids 1-111), p300 (amino acids 1-111), and β-catenin (amino acids 647-781) were expressed in E. coli and purified. β-catenin (5 μg) was bound to protein A-agarose beads coated with β-catenin-specific antibody (2.5 μg) and incubated with either CBP (5 μg) or p300 (5 μg). Unbound proteins were washed away, and specific interactions between these proteins were challenged by using 100 μM ICG-001. The complexes were washed and immunoblotted by using an anti-His antibody. (C) ICG-001 has no effect on AP1 and CRE reporter constructs. RLU, relative light units.

Fig. 3.

ICG-001 is specific for CBP but not p300. (A) Full-length CBP, p300, or β-catenin plasmids were cotransfected with TOPFLASH in SW480 cells. Luciferase assays were performed 24 h after ICG-001 treatment. (B) ICG-001 competes with β-catenin for CBP. SW480 cells were treated with ICG-001 or DMSO. Nuclear lysates were coimmunoprecipitated with anti-CBP or anti-p300 antibody and immunoblotted for β-catenin. (C) Nuclear lysates of treated C2C12 myoblasts (25 μM ICG-001, 24 h) were immunoprecipitated with anti-β-catenin antibody and immunoblotted with anti-CBP antibody (Upper, lanes 1 and 2) or anti-p300 antibody (lanes 3 and 4). ICG-001 does not affect levels of CBP, p300, or β-catenin (Lower). (D) SW480 cells were cotransfected with TOPFLASH and duplex siRNA against either CBP or p300. Twenty-four hours after transfection, the cells were treated with 10 μM ICG-001 or DMSO. Luciferase assays were performed 24 h later (Upper). Nuclear lysates from siRNA-transfected cells were immunoblotted (Lower).

Fig. 4.

ICG-001 inhibits the expression of the TCF/β-catenin-mediated genes survivin and cyclin D1. (A) Inhibition of survivin gene transcription was measured by semiquantitative RT-PCR in SW480 cells treated with ICG-001 (25 μM, 24 h) or DMSO. (B) Survivin immunoblots from SW480 and HCT116 cells treated with ICG-001 (10 and 25 μM, 24 h) or DMSO. (C) Cyclin D1 immunoblots from SW480 cells treated with 25 μM ICG-001 or DMSO. (D) Chromatin immunoprecipitation assay using the cyclin D1 promoter. After 8-h treatment with ICG-001 (25 μM) or DMSO, promoter occupancy was evaluated by using CBP-(AC-22) or p300-(C-20) specific antibodies.

Fig. 5.

ICG-001 is selectively cytotoxic to colon carcinoma cell lines and effective in vivo. (A) SW480, HCT116 cells and the normal colonic epithelial cells CCD-841Co were treated with ICG-001 (25 μM) or DMSO. Caspase 3/7 enzymatic activity was measured. Error bars represent mean ± SD. ^*, P < 0.05. (B) SW480 (▪), HCT116 (▴), and CCD-841Co (▾) cells were treated with increasing amounts of ICG-001, followed by MTS cell viability assay. (C) i.v administration (150 mg/kg) or vehicle (PBS) into 7-week-old female BALB/c nude mice for 22 days. Tumor growth inhibition (TGI) (Right) was calculated by using the following equation: % TGI = 100 × [1-(mean final tumor volume of treated group/mean fi-nal volume of vehicle group)]. Effect of treatment on body weight (Left). Error bars are mean ± SD; n = 10 for each treatment group; ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001. (D) Treated or vehicle-treated tumors were removed from xenografted animals and immunoblotted for Survivin.