Figure 6
Nuclear PAK4-LIFR Axis involved in breast-to-bone metastasis of ERα-positive breast cancer cells. a PAK4 regulates LIFR protein in breast cancer cells. ZR-75-30 cells were transiently transfected with control or increasing amounts of Flag-PAK4 expression plasmids with or without E2 (10-9 M) for 48 h. LIFR and PAK4 were detected by western blot analysis. GAPDH was used as a loading control. b MCF-7 cells infected with shPAK4 lentivirus or control shRNA (shCtrl) for 3 d and were either untreated or treated with E2 for 24 h. A western blot revealed PAK4 and LIFR protein levels. c PAK4 resulted in the suppression of LIFR gene expression. MCF-7 cells were transiently transfected with control or increasing amounts of Flag-PAK4 expression plasmid and treated with E2 (10−9 M) for 24 h. The cells were then harvested and analyzed for PAK4 and LIFR mRNA using quantitative real-time PCR assay. d MCF-7 cells infected with shPAK4 lentivirus or control shRNA (shCtrl) for 3 d and were either untreated or treated with E2 for 24 h. Real-time PCR assays revealed the PAK4 and LIFR mRNA levels. The real-time PCR values were normalized to the housekeeping gene β-actin. Experiments were performed three times, each with technical duplicates in the quantitative RT-PCR assay, and the data are presented as the means ± s.d. P < 0.05 according to Student’s t test. e, f PAK4 and ERα are predominantly recruited to the ERE of LIFR in the presence of E2 in MCF-7 cells. ChIP/re-ChIP experiments were performed using specific antibodies against PAK4 and ERα as indicated. g, h Number of invading cells (mean ± s.d.) from three indicated experiments in the presence of E2. **P < 0.01, ***P < 0.001 according to one-way ANOVA. i Schematic representation of PAK4 corepressor functions in ERα-induced transactivation
