Figure 5.

Ciclesonide reduced Yes-associated protein (YAP) nuclear localization and regulated breast CSC formation through YAP signaling. (A) After treatment with ciclesonide (10 μM), the transcript and protein levels of YAP1 were measured by real-time RT-qPCR using specific primers and an anti-YAP1 antibody, respectively. We used β-actin as the internal control. (B) Mammospheres were treated with ciclesonide (10 μM) for 1 day. After separation on 10% PAGE gels, cytosolic and nuclear protein extracts were transferred to membranes, followed by western blotting with anti-YAP and anti-p-YAP1 antibodies. β-actin and Lamin B were used as loading controls for the cytosolic and nuclear protein extracts, respectively. (C) siRNA-mediated silencing of GR decreased the expression of YAP and GR proteins in MCF-7 and MDA-MB-231 cells. GR protein expression was downregulated via GR siRNA, and the protein levels of YAP1 and GR were measured with anti-GR and anti-YAP1 antibodies. (D) siRNA-mediated silencing of YAP1 induced a significant decrease in the MFE in MDA-MB-231 cells. YAP1 was downregulated in MDA-MB-231 cells by YAP1 siRNA, and a mammosphere formation assay was performed with these cells. The experiment was conducted with cancer cells treated with ciclesonide in a single passage of spheres formation assay. (E) Treatment with verteporfin, a suppressor of YAP-TEAD complex formation, resulted in a decrease in the MFE in MDA-MB-231 cells. The cancer cells were cultured in a 96-well plate with the indicated concentration of verteporfin. The proliferation of the cells was measured with MTS reagent. Data from triplicate experiments are shown as the means ± SDs; * p < 0.05 vs. the control. Experiments were repeated three times. Mammospheres were cultured for 7 days in MammoCult medium. After treatment with verteporfin (2 and 5 μM), mammosphere formation was evaluated. The experiment was conducted with cancer cells treated with ciclesonide in a single passage of spheres formation assay. * p < 0.05 vs. the control. Experiments were repeated three times. (F) After treatment with ciclesonide, RT-qPCR of mammospheres was performed using CTGF- and CYR61-specific primers. Data from triplicate experiments are shown as the means ± SDs; * p < 0.05 vs. the control. Experiments were repeated three times. (G) Correlation analysis of YAP1 and GR (NR3C1 gene) in breast cancer patients from a publicly available TCGA dataset. Analysis of the breast invasive carcinoma dataset showed that the correlation coefficient of YAP1 and GR (NR3C1 gene) was 0.5. p-value = 3.2 × 10⁻⁷⁰.