Fig. 9: IWR-1 Axin1 stabilizer mimics p140Cap ability to improve the destruction complex stability and to curb tumor progression.

a Effect of IWR-1 on the destruction complex stability. Immunoprecipitation of β-Catenin from Mock TuBo mammospheres treated for 5 consecutive days with 1 μM IWR-1 or DMSO (control vehicle). Immunoblot with indicated antibodies. GAPDH was used as loading control (n = 3). Data are representative of n = 3 experimental repeats. b Effect of IWR-1 treatment on mammosphere formation efficiency. Mammosphere formation assay from untreated Mock and p140Cap cells or Mock TuBo mammosphere treated with IWR-1 as in (a). Bar plots of mammosphere numbers are shown. Data are represented for n = 4 experimental repeats/group, as mean ± SEM; two-tailed unpaired t test. c G-CSF protein levels in mammosphere supernatants, treated as in (a). G-CSF protein levels were measured by ELISA in supernatants from untreated Mock and p140Cap or Mock TuBo mammospheres treated with IWR-1 as in (a). Data are represented for n = 3 experimental repeats/group, as mean ± SEM; two-tailed unpaired t test. d Immunoblot analysis of active β-Catenin in untreated Mock and p140Cap cells, or Mock TuBo mammospheres treated with IWR-1 as in (a). GAPDH was used as loading control. Data are representative of n = 5 experimental repeats and shown as mean ± SEM; two-tailed unpaired t test. e 10⁵ cells were injected into the mammary fat pad of 6-weeks-old BALB/C mice, tumor growth was monitored and tumor size was measured. Mock TuBo tumor-bearing mice were treated with IWR-1 (n = 5) (5 mg/Kg) every two days, with six different injections, starting when the tumor size had reached approximately 80 mm³. Mock (n = 6) and p140Cap TuBo (n = 6) tumor-bearing mice were treated with DMSO as controls. Data are represented for n = x mice and shown as mean ± SEM; 2way ANOVA. f Representative flow cytometry bar plots show the percentage of PMN-MDSCs cells normalized on CD45⁺ cells, in tumor, blood and spleen of mice described in (e). TuBo vehicle (n = 7/tumor, n = 5/blood, n = 6/spleen); TuBo p140Cap vehicle (n = 6/tumor, n = 5/blood, n = 5/spleen); TuBo IWR-1 (n = 7/tumor, n = 6/blood, n = 6/spleen). Data are represented for n = x mice and shown as mean ± SEM; two-tailed unpaired t test. g G-CSF levels in sera of IWR-treated or untreated tumor-bearing mice as in (e), by ELISA. TuBo vehicle (n = 6); TuBo p140Cap vehicle (n = 4); TuBo IWR-1 (n = 6). Data are represented for n = x mice and shown as mean ± SEM; two-tailed unpaired t test.