Figure 1.
IHC staining of ZNF703 in 127 cases of breast cancer tissue microarrays.
(A) Immunohistochemistry (IHC) scoring of ZNF703 in 127 breast cancer patients. The vertical axis indicates the difference between the score of each patient and the median score. The dotted line represents the cut-off point by which patients were divided into high and low ZNF703 expression groups. (B) Representative image of IHC staining for ZNF703 in breast cancer specimens. Left: low ZNF703 expression; Right: high ZNF703 expression. The bar represents 10 µm. (C) High ZNF703 expression was mainly present in ER+ and PR+ breast cancers. Left: proportion of patients with ER+ or ER− breast cancer in the high or low ZNF703 expression groups, respectively. Right: proportion of patients with PR+ or PR− breast cancer in the high or low ZNF703 expression groups, respectively. Also see Table S1.
Figure 2.
Breast cancer cell lines with high ZNF703 expression have different modes of tamoxifen resistance.
(A) RT-PCR was used to evaluate ZNF703 mRNA expression levels in 11 breast cancer cell lines and the normal breast epithelial cell line MCF-10A. (B, C) MCF-7 and BT-549 cells were treated with DMSO or different concentrations of TAM, with the addition of 1 nM estradiol in each well. Inhibitory rates were examined using the SRB cell growth assay after 4 days. (D) Similar to MCF-7 cells, HCC1500 cells were treated with DMSO or different concentrations of TAM. The arrow shows where the inhibitory rate was less than 30% after treatment with 1 µM TAM. (E) MDA-MB-134 cells were treated with DMSO or different concentrations of TAM. The double-arrow shows where the inhibitory rate was 80% after treatment with 100 µM TAM. The single-arrow indicates that 10−2 µM or 10−3 µM TAM stimulated growth of MDA-MB-134 cells. Data are representative of three experiments and are presented as mean ± SD.
Figure 3.
Overexpression of ZNF703 activates p-AKT and induces tamoxifen resistance.
(A) MCF-7-vector and MCF-7-ZNF703 cells were treated with DMSO, 1 µM, 2 µM or 5 µM TAM for 24 hours, followed by western blot analysis of the indicated proteins. GAPDH was used as a loading control. ImageJ software was used to measure p-AKT levels and normalize to GAPDH levels (y-axis). (B) Western blot analysis of ZNF703 protein expression in MCF-7-vector and MCF-7-ZNF703 cells. MCF-7-vector and MCF-7-ZNF703 cells were treated with DMSO or different concentrations of TAM for 72 hours and inhibitory rates were examined. (C) Tamoxifen-mediated cell growth inhibition in MCF-7-vector and MCF-7-ZNF703 cells was evaluated by the colony formation assay. MCF-7-vector and MCF-7-ZNF703 cells were treated with DMSO or 1 µM TAM for 15 days, and colony forming areas were calculated to compare the differences before and after administration in both cell lines. Data are representative of three independent experiments and are presented as mean ± SD. *P<0.05, **P<0.01.
Figure 4.
Increasing effects of TAM in MDA-MB-134 and HCC1500 cells after ZNF703 knockdown.
(A) MCF-7-ZNF703 cells were treated with DMSO or different concentrations of TAM for 3 or 6 days, and the optical density (OD) was measured by the SRB assay. The y-axis represents the ratio of OD in TAM-treated cells compared to DMSO-treated cells. (B) Western blot analysis of ZNF703 and GAPDH in MDA-MB-134 non-targeting siRNA control (NC), MDA-MB-134 siRNA-1 or MDA-MB-134 siRNA-2 cells. Cells were treated with DMSO or different concentration of TAM for 6 days, and survival rates were examined. MDA-MB-134 NC vs. MDA-MB-134 siRNA-1 cells, P<0.05; MDA-MB-134 NC vs. MDA-MB-134 siRNA-2 cells, P<0.05; MDA-MB-134 siRNA-1 vs. MDA-MB-134 siRNA-2 cells, P>0.05. Statistical analyses were done by paired t-test. (C) Western blot analysis of ZNF703 and GAPDH in HCC1500 NC, HCC1500 siRNA-1, and HCC1500 siRNA-2 cells. Cells were treated with DMSO or different concentrations of TAM for 6 days, and survival rates were examined. HCC1500 NC vs. HCC1500 siRNA-1 cells, P<0.05; HCC1500 NC vs. HCC1500 siRNA-2 cells, P<0.05; HCC1500 siRNA-1 vs. HCC1500 siRNA-2 cells, P<0.05. Data is representative of at least three independent experiments and are presented as mean ± SD. *P<0.05, **P<0.01.
Figure 5.
ZNF703-mediated activation of Akt/mTOR signaling can be inhibited by rapamycin.
(A) MCF-7-vector and MCF-7-ZNF703 cells were treated with DMSO, 0.1 µM rapamycin, or 1 µM rapamycin for 24 hours before cell lysis. Western blot analysis of lysates using the indicated antibodies. A GAPDH antibody was used as a loading control (B) MCF-7-vector and MCF-7-ZNF703 cells were treated with DMSO, 0.01 µM rapamycin, or 0.1 µM rapamycin for 24 hours. Western blot analysis using the indicated antibodies. (C) DMSO or various concentrations of TAM were added to three MCF-7-ZNF703 cell groups that were treated with DMSO, 0.1 µM rapamycin, or 1 µM rapamycin for 72 hours. Survival rates were examined by the SRB assay. MCF-7-ZNF703 DMSO vs. MCF-7-ZNF703 0.1 µM rapamycin, P<0.001; MCF-7-ZNF703 DMSO vs. MCF-7-ZNF703 1 µM rapamycin, P<0.01; MCF-7-ZNF703 0.1 µM rapamycin vs. MCF-7-ZNF703 1 µM rapamycin, P>0.05, paired t-test was performed. (D) Expression of p-mTOR (S2448), p-4E-BP1 (T37/46) and p-4E-BP1 (T70) in (B) were measured using ImageJ software and normalized to GAPDH levels (y-axis). *P<0.05, **P<0.01.