siWNT4-mediated growth suppression is mediated by increased CDKN1A/p21. a Breast cancer cell lines (BCCLs) were reverse-transfected with 10 nM small interfering RNA (siRNA), and RNA was collected 48 h posttransfection. Gene expression data are shown as the mean of biological triplicate ± SD. p < 0.05 for each SCR vs WNT4 (t test). b
Left, SUM44PE (44PE) cells were reverse-transfected with 10 nM siRNA, and lysates were collected 48 h posttransfection. The samples presented were also used in Fig. 5d, and the loading control is replicated for clarity. Data are representative of duplicate experiments. Right, MDA-MB-134-VI (MM134) cells were reverse-transfected with 10 nM siRNA (20 nM total for combinations), and lysates were collected 48 h posttransfection. 1, GE Dharmacon siGENOME control pool 2; 2, Sigma-Aldrich MISSION control pool 1. Reduction in p21 protein levels with scrambled control siRNA was a class effect across all commercial scrambled siRNA pools tested, observed only in MM134 cells. c
Left, 44PE cells were reverse-transfected with increasing concentrations of siWNT4 in the presence of 10 nM siSCR or siCDKN1A. Proliferation was assessed 7 days posttransfection. Data are shown as fold change vs siSCR or siCDKN1A control (no siWNT4). p < 0.01 by analysis of variance (ANOVA) for interaction (siSCR vs siCDKN1A on siWNT4 effect). Right, MM134 cells were reverse-transfected with 10 nM siRNA as indicated. Proliferation was assessed 7 days posttransfection. Data are shown as fold change vs mock transfection. *p < 0.05 by ANOVA (Dunnett’s multiple comparisons test) vs siWNT4 alone. n.s. Not significant. d Schematic of WNT4 regulation and signaling in invasive lobular carcinoma (ILC) and ILC endocrine-resistant long-term estrogen-deprived (ILC-LTED) cells. Red stars, WNT4 estrogen receptor binding site. Left, in parental ILC cells, 17β-estradiol (E2) activates the estrogen receptor (ER), which binds at WNT4 and drives WNT4 expression. Center, in 134:LTED, the ER no longer binds at WNT4, and activated nuclear factor kB (NF-kB) drives increased WNT4 expression. Right, In 44:LTED, the ER binds at WNT4 despite the absence of exogenous ligands and, potentially in coordination with Oct-4, maintains weaker WNT4 expression. Bottom, WNT4 initiates a Wnt signaling pathway that is likely β-catenin-independent. This leads to suppression of CDKN1A expression and a decrease in p21 protein, which relieves p21-mediated growth inhibition and permits cell growth.