Fig. 3
Disrupting ATO binding to Pin1 impairs its ability to induce Pin1 degradation and to inhibit breast cancer tumor growth. a The co-crystal structure of ATO and Pin1 complexes suggests that the M130V, but not M130I Pin1 mutation, would prevent ATO from binding to the Pin1 active site (arrows). b Biotin-ATO binds to the M130I, but not M130V Pin1 mutant. Pin1 and its mutants were incubated with different concentrations of biotinylated ATO, followed by isolating biotin-ATO-bound Pin1 or its mutants using NeutrAvidin beads. ATO-bound Pin1 were detected by immunoblot and plotted against ATO concentrations. c–f The M130V, but not M130I, Pin1 mutation impairs ATO’s ability to induce Pin1 degradation and inhibit cell growth. Pin1 CRISPR cells stably expressing Pin1 or its M130V or M130I mutant (c) were treated with ATO, followed by assaying Pin1 levels (d, e) or cell growth (f). g–j The M130V, but not M130I Pin1, mutation impairs the ability of ATO to inhibit tumor growth in mice. Female nude mice were flank inoculated with 1 × 106 Pin1 CRISPR cells that stably re-expressed Pin1, or its M130V or M130I mutant, and 1 week later, treated with ATO (2 mg/kg, i.p., 3 times/week). Tumor sizes were weekly measured (g) and mice were sacrificed after 5 weeks to collect tumor tissues (h) and measure their weights (i), as well as their expression of Pin1 and selected Pin1 substrate (j). The results are expressed as mean ± S.D. and the P values (*P < 0.05, **P < 0.01, ***P < 0.001) were determined by ANOVA test. n = 4–6 mice
