Article Figures & Data
Figures
- Figure 1.
PELP1 is needed for optimal activation of the mTOR pathway. A and B, MCF7 and ZR75 cells were transiently transfected with nonspecific control siRNA or PELP1-specific siRNA for 72 hours, serum starved for 24 hours, and stimulated with 10% serum for 10 minutes. Status of phosphorylation of mTOR signaling components was analyzed by Western blotting. Quantitation of phosphorylation after normalizing to its respective total protein is shown. C, lysates of MCF7 breast cancer cells were subjected to immunoprecipitation with PELP1, Raptor, and Rictor antibodies, and the presence of mTOR and PELP1 in the immunoprecipitates was analyzed by Western blotting.
- Figure 2.
mTOR inhibitors reduced PELP1-mediated signaling and cell proliferation. A, cell viability of MCF7-vec and MCF7-PELP1#20 cells was analyzed after treating the cells with or without 20 nmol/L of rapamycin (Rapa) or AZD8055 in 5% FBS in RPMI medium for 72 hours using an MTT assay. Results are the mean value of experiment performed in triplicate. B, cell viability of MCF7-vec, and MCF7-PELP1#13 cells was analyzed after treating the cells with or without 20 nmol/L of rapamycin or AZD8055 in 5% DCC serum in RPMI medium for 72 hours using MTT assay. Results are the mean value of experiments performed in triplicate. C and D, model cells were plated on cover slips in 6-well plates, treated with or without 40 nmol/L of rapamycin or 20 nmol/L AZD8055 in 5% DCC serum in RPMI medium. C, Ki-67 staining as a marker of proliferation was performed as described in methods section. D, TUNEL staining was performed as a marker of apoptosis on fixed cells. Quantitation of Ki-67 and TUNEL staining was done as described in Materials and Methods. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
- Figure 3.
mTOR inhibitors reduced PELP1-mediated estrogen-driven growth in breast cancer cells. MCF7 (A) or ZR75 (B) cells (2 × 103) stably expressing control vector or PELP1 WT were seeded in 96-well plates and stimulated with E2 (1 × 10−8 mol/L) in 5% DCC medium for 7 days in the presence or absence of mTOR inhibitors. Cell viability was determined by using an MTT assay. Results are the mean value of experiments performed in triplicate. Student t test was used for analysis. **, P < 0.01; ***, P < 0.001. C, ZR75 cells stably expressing control shRNA or PELP1 shRNA were E2 starved for 72 hours and stimulated with E2 (1 × 10−8 mol/L) for 15 minutes in 5% DCC media. Status of mTOR signaling was analyzed by Western blot analysis. Quantitation of phosphorylation after normalizing to its respective total protein is shown.
- Figure 4.
Rapamycin (Rapa) or AZD8055 treatment reduced PELP1-mediated xenograft tumor growth. A, six-week-old nude female mice were subcutaneously implanted with MCF7-PELP1 WT cells. After the tumors reached a measurable size, the mice were treated daily with vehicle, rapamycin, or AZD8055 for 4 weeks. Tumor volumes were measured at weekly intervals. B, tumor weights are shown in the histogram. C, Ki-67 expression was analyzed by using immunohistochemistry and quantitation of Ki-67 staining was done as described in the Materials and Methods section. D, TUNEL staining was performed as a marker of apoptosis on tumors that were treated with rapamycin or AZD8055. Quantitation was done as described in Materials and Methods. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
- Figure 5.
PELP1 cytoplasmic localization promotes mTOR signaling and tamoxifen resistance. A, MCF7 cells stably expressing control vector or PELP1cyto were serum starved for 72 hours and stimulated with 10% serum for 10 minutes. The status of PELP1 expression and S6 phosphorylation was analyzed by Western blotting. Quantitation of S6 phosphorylation after normalizing to its respective total protein is shown. B, MCF7 control vector, MCF7-PELP1cyto, or MCF7-PELP1-WT cells were treated with vehicle or 2.5 μmol/L tamoxifen for 72 hours in 5% DCC medium and cell viability was assayed by using an MTT assay. C, MCF7 control vector or MCF7-PELP1cyto cells were treated with vehicle, 2.5 μmol/L tamoxifen, 20 nmol/L rapamycin (Rapa), or in combination for 7 days in 5% DCC medium. After the treatment, cell viability was determined by using an MTT assay. D, total lysates from various model cells were analyzed for the expression of PELP1 by Western blotting. E, MCF7-TamR cells were treated with vehicle, 2.5 μmol/L tamoxifen, 20 nmol/L rapamycin, or in combination for 7 days, and cell viability was determined by using an MTT assay. F, MCF7-HER2 cells were treated with or without 2.5 μmol/L tamoxifen, 20 nmol/L rapamycin, or in combination for 7 days, and cell viability was determined by using an MTT assay. Student t test was used to analyze the data. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
- Figure 6.
mTOR inhibitors reduce the PELP1–tamoxifen-mediated subcutaneous xenograft tumor growth. A, six-week-old nude female mice were subcutaneously implanted with MCF7-PELP1cyto cells. After the tumors reached a measurable size, the mice were treated daily with vehicle, tamoxifen alone, or tamoxifen and AZD8055 together for 4 weeks. Tumor volumes were measured at weekly intervals. B, tumor weights are shown in the histogram. C, Ki-67 expression was analyzed by using immunohistochemistry, and quantitation was done as described in Materials and Methods section. D, TUNEL staining was performed as a marker of apoptosis on tumors. Quantitation was done as described in Materials and Methods. *, P < 0.05;***, P < 0.001; ****, P < 0.0001.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure 1 - PDF - 226K, PELP1 down regulation or upregulation alters mTOR signaling and PELP1 interacts with mTOR.
- Supplementary Figure 2 - PDF - 115K, mTOR inhibitors reduce proliferation and promote apoptosis of PELP1-WT driven tumors.
- Supplementary Figure 3 - PDF - 106K, Synergistic effect of mTOR inhibitor and tamoxifen on therapy resistant cells.
- Supplementary Figure 4 - PDF 385K, mTOR inhibitors reduce the tamoxifen driven PELP1-mediated subcutaneous xenograft tumor growth.
Volume 13, Issue 6
