Inhibition of MEK1/2 signaling attenuates Six1-induced increases in TICs. (A) Inhibition of MEK1/2 with U0126 reduces the CD24low CD44+ TIC population. Cells were treated with10 μM U0126 for ten days. The graph represents an average of at least three experiments with three clones. Error bars represent mean +/- SEM. P values calculated using a two-tailed t test. (B) Inhibition of MEK1/2 with U0126 reduces secondary tumorsphere formation in MCF7-Six1 cells. Representative secondary tumorsphere assays at d10. The cells were treated with 10 μM U0126 throughout the assay. Experiments were performed at least three times. Error bars represent mean +/- SEM. P values calculated using a two-tailed t test. (C) Inhibition of MEK1/2 reverses β-catenin dependent transcription, as measured using a β-catenin responsive promoter (Top-flash) after normalization to renilla luciferase. Experiments were performed at least three times. Error bars represent mean +/- SEM. P values calculated using a two-tailed t test. (D) Inhibition of MEK1/2 by AZD6244 decreases tumor formation efficiency in NOD/SCID mice. A total of 104 MCF7-Six1 or MCF7-Ctrl cells (three clones each) were injected underneath the nipple of the fourth mammary gland of six-week old female NOD/SCID mice. One week post injection, the mice were treated with AZD6244 by oral gavage twice per day for three days and once/day for the following three days (vehicle, 25 mg/kg or 50 mg/kg) and were monitored weekly for tumor formation (five week data shown). Statistical analysis: Extreme Limiting Dilution Analysis. Six1 with vehicle versus Six1 with AZD6244 25 mg/kg, P < 0.05; Six1 with vehicle versus Six1 with AZD6244 50 mg/kg, P < 0.005. ERK, extracellular growth factor receptor; MEK, mitogen activated protein kinase; SEM, standard error of the mean; TIC, tumor initiating cell.