Figure 1.
Tamoxifen induces ER-α36, HER2 and EGFR expression.
Western blot analysis of the expression levels of ER-α66, ER-α36, HER2 and EGFR in ER-positive breast cancer MCF7 (A), T47D (B) and H3396 (C) cells treated with 1 µM of tamoxifen (TAM) for indicated time period.
Figure 2.
Tamoxifen induces ER-α36, HER2 and EGFR expression via the ER-α36-EGFR/HER2 positive regulatory loops.
A. Western blot analysis of the expression levels of ER-α66, ER-α36, HER2 and EGFR in MCF7 cells with knocked-down levels of ER-α36 expression (MCF7/Si36) treated with 1 µM of TAM for indicated time period. B. ER-α66, ER-α36, HER2 and EGFR expression in MCF7 cells treated with 1 µM of TAM together with 1 µM of Broussoflavonol B (BB) for indicated time period. C. Western blot analysis of ER-α66, ER-α36, HER2 and EGFR in MCF7 cells treated with 1 µM of TAM and 1 µM of Lapatinib for indicated time period.
Figure 3.
Tamoxifen resistant ER-positive breast cancer MCF7 cells express ER-α36-EGFR/HER2 regulatory loops.
A. ER-positive breast cancer MCF7 cells and tamoxifen resistant MCF7 cells (MCF7/TAM) cells were treated with indicated concentrations of TAM for seven days and survived cells were counted. Each point represents the means of three experiments; bars, SE. B. Western blot analysis of the expression levels of ER-α66, ER-α36, EGFR and HER2 in parental MCF7 (MCF7/P) and MCF7/TAM cells. C. Expression of ER-α66, ER-α36, EGFR and HER2 in MCF7/TAM cells transfected with the empty expression vector (MCF7/TAM/V) and MCF7/TAM cells with knocked-down levels of ER-α36 expression (MCF7/TAM/Si36). D. Expression of ER-α66, ER-α36, EGFR and HER2 in MCF7 cells transfected with the empty expression vector (MCF7/V) and MCF7 cells with forced expression of ER-α36 recombinant DNA (MCF7/ER36).
Figure 4.
Dual kinase inhibitor Lapatinib downregulates ER-α36 expression and sensitizes MCF7/TAM cells to tamoxifen.
A. Western blot analysis of the expression of ER-α36 and 66 as well as EGFR and HER2, and levels of EGFR and HER2 phosphorylation in parental MCF7 (MCF7/P) and MCF7/TAM cells treated with indicated concentrations of Lapatinib for 12 hours. B. Cells were treated with indicated concentrations of TAM together with vehicle or 1 µM of Lapatinib (LAP) for seven days and the numbers of survived cells were counted. The columns represent the means of three experiments; bars, SE. *, P<0.05 for MCF/TAM cells treated with vehicle vs cells treated with 1 µM of tamoxifen and Lapatinib.
Figure 5.
ER-α36 disruptor Broussoflavonol B restores TAM sensitivity.
A. Western blot analysis of the expression of ER-α36, ER-α66, EGFR and HER2, and levels of EGFR and HER2 phosphorylation in parental MCF7 (MCF7/P) and MCF7/TAM cells treated with indicated concentrations of Broussoflavonol B (BB) for 12 hours. B. Cells were treated with indicated concentrations of tamoxifen (TAM) together with vehicle or 1 µM of Broussoflavonol B (BB) for seven days and the numbers of survived cells were counted. The columns represent the means of three experiments; bars, SE. *, P<0.05 for MCF/TAM cells treated with vehicle vs cells treated with 1 µM of tamoxifen and BB.
Figure 6.
Tumorsphere cells derived from ER-positive breast cancer MCF7 cells express ER-α36-EGFR/HER2 positive regulatory loops.
A. Western blot analysis of the expression of ER-α36, ER-α66, EGFR, HER2 and ALDH1 in the monolayer MCF7 cells grown on attachment dishes (MCF7/P) and MCF7 tumorsphere cells grown on low-attachment dishes. B. Tumorsphere cells derived from MCF7TAM cells were treated with 5 µM of Broussoflavonol B (BB) or Lapatinib (LAP) for five days. Western blot analysis of expression levels of different proteins was performed. C. Tumorsphere cells derived from MCF7TAM cells were treated with 2.5 µM of Broussoflavonol B (BB), Lapatinib (LAP) or BB (2.5 µM) and LAP (2.5 µM) together for five days. Western blot analysis of expression levels of different proteins was performed.
Figure 7.
Disruption of the ER-α36-EGFR/HER2 positive regulatory loops sensitizes ER-positive breast cancer stem/progenitor cells to TAM.
A. Tumorsphere formation assay was used to assess the effects of TAM alone or together with Lapatinib (LAP) or Broussoflavonol B (BB) on ER-positive breast cancer stem/progenitor cells derived from parental MCF7 (MCF7/P) and tamoxifen resistant MCF7 cells (MCF7/TAM). The representative results are shown. B. The numbers of tumorspheres formed by MCF7/P and MCF7/TAM cells in the presence or absence of LAP and BB for 1st and 2nd generations. C. The number of cells from dissociated tumorspheres formed by MCF7/P and MCF7/TAM cells in the presence or absence of LAP and BB for 1st and 2nd generations. The columns represent the means of three experiments; bars, SE. * & #, P<0.05 for MCF/TAM cells treated with vehicle vs cells treated with tamoxifen and LAP or BB, respectively.
Figure 8.
Combination of Broussoflavonol B and Lapatinib sensitizes ER-positive breast cancer stem/progenitor cells to TAM.
A. Tumorsphere formation assay was used to assess the effects of TAM alone or together with 2.5 µM of Lapatinib (LAP), Broussoflavonol B (BB) or BB (2.5 µM) and LAP (2.5 µM) on ER-positive breast cancer stem/progenitor cells derived from parental MCF7 (MCF7/P) and tamoxifen resistant MCF7 cells (MCF7/TAM). The representative results are shown. B. The numbers of tumorspheres formed by MCF7/P and MCF7/TAM cells in the absence or presence of LAP (2.5 µM), BB (2.5 µM) or LAP (2.5 µM) and BB (2.5 µM) together. C. The number of cells from dissociated tumorspheres formed by MCF7/P and MCF7/TAM cells in the absence or presence of LAP (2.5 µM), BB (2.5 µM) or LAP (2.5 µM) and BB (2.5 µM) together. The columns represent the means of three experiments; bars, SE. *, # and Δ, P<0.05 for MCF/TAM cells treated with vehicle vs cells treated with tamoxifen and LAP, BB, or LAP+BB together.