Fig. 2
SRCs are key co-regulators of WT, Y537S, and D538G ERα transcriptional activity and are essential for breast cancer cell growth. a Knockdown of SRC-3 by co-transfection of HeLa cells with an siRNA targeting pool reduced transcriptional activity of LBD point mutant ERα proteins, as assayed by an ERE-luciferase reporter, in hormone-depleted media. Data are represented as mean ± SEM (n = 3); ***p < 0.001. RLU relative light units. b A “pan-SRC” inhibitor, SI-1, reduced all three SRC protein levels in MCF-7 breast cancer cells treated overnight, as assayed by immunoblotting. β-actin serves as a loading control. c SRC inhibitor SI-1 reduced WT, Y537S, and D538G ERα transcriptional activity at concentrations higher than the established IC50. Vectors expressing YFP-tagged WT, Y537S, and D538G ERα proteins were co-expressed with pERE-E1b-luc in HeLa cells, and then cells were treated with dimethyl sulphoxide (DMSO, vehicle control) or SI-1 overnight. Luciferase activity was measured as in Fig. 1b. Data are represented as mean ± SEM (n = 3). d,e Combination of an SRC inhibitor (SI-1) and an oral SERD (AZD9496) synergistically reduce Y537S (d) and D538G (e) mutant ERα transcriptional activity. Experiment was done as in (c), except that AZD9496 was added with or without SI-1 to co-transfected HeLa cells. Data are represented as mean (n = 3). f The oral SERD AZD9496 is more effective than the SRC inhibitor SI-1 in reducing cell viability of MCF-7 lines expressing WT or Y537S ERα. The lentiviral transduced MCF-7 stably expressing cell lines were treated with vehicle (DMSO) or different concentrations of AZD9496 or SI-1 as indicated. After 6 days of treatment, viability was assayed by a MTT assay. Data are represented as mean ± SEM (n = 3). Synergism was observed with combination (Combo) treatments 4 and 5 (12.5/200 nM AZD9496/SI-1; 25/400 nM AZD9496/SI-1) in Y537S ERα-expressing cells (shown as red arrows; CI values are shown in Supplementary Table 5)
