Fig. 4: Resistant cells regain sensitivity to tamoxifen and letrozole following MCM3 knockdown.

Increased MCM3 protein levels in tamoxifen- or aromatase inhibitor-resistant vs. -sensitive cell lines was confirmed by Western blotting using whole cell lysate in (a) two tamoxifen-resistant models, one MCF-7-based (MCF-7/S0.5 vs. TamR-1, TamR-4 and TamR-7) and one T47D-based (T47D/S2 vs. T47D/TR-1 and T47D/TR-2) and in (b) a letrozole-resistant model (MCF-7/S0.5 vs. LetR1). Transfection of cells with 2 separate MCM3 targeting siRNAs (siMCM3.2 and siMCM3.6) resulted in > 75% reduction in the expression of MCM3 compared to a scrambled siRNA (siControl) used as a control, as measured by RT-qPCR at 48 h (c) and Western blotting at 96 h (d) or at 48, 72, 96 and 120 h (e). Data are representive of three independent experiments. (f) Knockdown of MCM3 by either of the two siRNAs in TamR cells treated with 10⁻⁶M tamoxifen significantly decreased growth compared to siControl transfected cells measured by a colorimetric crystal violet assay at 120 h. The reduction in cell growth of MCM3 knockdown was confirmed in three independent experiments. Data is represented as OD590 values ± s.e.m of triplicates. (g) Cell proliferation as measured by BrdU incorporation demonstrated significantly lower proliferation of TamR-1 cells transfected with siMCM3.2 and siMCM3.6 compared to siControl-transfected cells. Similarly, transfection of the tamoxifen-resistant cell lines T47D/S2, T47D/TR-1, and T47D/TR-2 with two separate siRNAs targeting MCM3 lead to 70–90% reduction of MCM3 levels vs. siControl, as measured by RT-qPCR at 48 h (c) and by Western blotting at 96 h (h), and significantly reduced growth of T47D/TR-1 and T47D/TR-2 in the presence of 10⁻⁶M tamoxifen (i) as measured by a colorimetric crystal violet assay. In contrast, the growth of the tamoxifen-sensitive parental cell lines MCF-7/S0.5 (f) and T47D/S.2 (i) or tamoxifen-resistant TamR-1 cells cultured in the absence of tamoxifen (j) transfected with siMCM3 was not significantly reduced compared to cells transfected with siControl. A representative of three independent experiments in which data is represented by OD590 values ± s.e.m in triplicates is shown, *p < 0.05. Transfection of the letrozole-resistant cell line LetR1 vs. MCF-7/S0.5 with two separate siRNAs targeting MCM3 resulted in significant reduction of MCM3 levels vs. siControl as measured by Western blotting at 96 h (k), and significantly reduced growth of LetR1 in the presence, but not in the absence, of letrozole (l), as measured by a colorimetric crystal violet assay at 120 h. *p < 0.05. PUM1 was used as a reference gene in the RT-qPCR and β-actin or GAPDH was used as loading control for Western blotting.