Fig. 9: PSMD14 inhibition could restore tamoxifen sensitivity in endocrine resistant breast cancer model.

A PSMD14 depletion decreases ERα protein level and ERα Y537S level in MCF-7 Y537S cells. Cell lysates were immunoblotted with the indicated antibodies. β-Actin was used as internal control. B PSMD14 depletion sensitizes tamoxifen inhibition effect in MCF-7 Y537S cells. MCF-7 Y537S cells were transfected with siPSMD14 or siControl. After 48 h, cells were plated into 96-well plate, while each well contained 5000 cells. The indicated tamoxifen concentrations were used for 48 h. The numbers of the cells were determined via CCK8 kit for the cellar metabolic activity. Experiments were done in triplicates. *P < 0.05; **P < 0.01; ***P < 0.001 for cell growth comparison. C PSMD14 depletion could restore the inhibition effect of tamoxifen in ERα target genes. MCF-7 Y537S cells in charcoal-stripped FBS and phenol red-free DMEM were transfected with siControl or siPSMD14 for 24 h. Then treated with 1 μM Tamoxifen for 12 h. Total RNA was extracted for gene expression analysis. Each group was analyzed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 for target gene expression comparison. D Luciferase assays showing PSMD14 depletion could restore the inhibition effect of tamoxifen in ERE-luciferase activity in MCF-7 Y537S cells. E PSMD14 depletion could restore the inhibition effect of tamoxifen in MCF Y537S cells. MCF-7 Y537S cells in charcoal-stripped FBS and phenol red-free DMEM were transfected with siControl or siPSMD14 for 24 h. Then treated with 1 μM Tamoxifen for 12 h. Then a CCK-8 assay was used to determine the cellular metabolic activity at the indicated time points after Thiolutin treated. Experiments were performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 for cell growth comparisons. F, G PSMD14 depletion could reduce the number of EdU-positive cells in MCF Y537S cells. MCF-7 Y537S cells in charcoal-stripped FBS and phenol red-free DMEM were transfected with siControl or siPSMD14 for 24 h. Then treated with 1 μM Tamoxifen for 12 h. Then EdU was added to the medium for 2 h of incubation. The absolute cell number was determined to indicate cell proliferation activity. Right panel shows quantification of Edu results by ImageJ software. Scale bar 100 μm. N = 3, *P < 0.05; **P < 0.01; ***P < 0.001 for cell growth comparisons. H, I Cell-cycle analysis by flow cytometry of MCF-7 Y537S cells were transfected with siControl or siPSMD14 for 24 h. Then treated with 1 μM Tamoxifen for 12 h. Then the cells were harvested, fixed with 70% ethanol, and stained with propidium iodide. The cells were subjected to FACS analysis. Experiments were performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 for cell proportion comparisons. PSMD14 depletion could not only impair breast tumor growth but also could strengthen the inhibition effect by tamoxifen in Y537S-expression MCF-7 cells by a xenograft model. Harvested and photographed tumors in the shPSMD14 and the Control group (J), tumor volume (K) and weight (L) growth in each mouse from the shPSMD14 group and the Control group in vivo. Data are shown as mean ± SD. Two tailed t test. *P < 0.05; **P < 0.01; ***P < 0.001.