Fig. 2
Pharmacological blockade of CDK2-EZH2 axis switches tumor cell lineage. a Murine basal-like cell line 4T1 cells were orthotopically transplanted on the Balb/c female mice, then treated with either vehicle or GSK343 at 100 mg kg−1 orally daily for 9 days. Tumor volume was measured. Representative tumor samples were displayed. Points represent mean ± SEM. Data was analyzed by two-tailed Student’s t test: **p < 0.05. b Whole tissue lysates from different tumors treated with either vehicle (Veh1 and Veh2) or GSK343 (GSK1, GSK2, and GSK3) were subjected to western blot analysis. Cell lysates from luminal T47D cells and T47D-expressing EZH2T416D cells (basal-T416D) were added as positive and negative control, respectively, for GATA3 and ERα. Quantitation analysis of the images of experiments performed by using ImageQuant TL Toolbox v8.1 and normalized with loading control actin. The results are presented as bar graphs shown below. c Under 3D-Matrigel culture conditions, MDA-MB-231 cells were treated with different concentrations of EZH2 inhibitor, EPZ-6438 (EPZi) as indicated. Whole-cell lysates were subjected to western blot to detect ERα and H3K27me3. d, e MDA-MB-231 cells in Matrigel 3D culture were treated with dinaciclib (DINA) at different concentrations for 3 days. Whole-cell lysates of the treated cells were immunoblotted with specific antibodies as indicated. pT416-EZH2 (p-EZH2) was probed using specific monoclonal antibody d. Activated CDK2 was detected with antibody against pCDK2 (T160) e. f Different TNBC cell lines in 3D culture were treated with DINA at 50 nM for 48 hours and whole-cell lysates were used for immunoblot with indicated antibodies. g MDA-MB-231 and BT 549 in 3D-Matrigel culture were treated with different CDK2i, SNS032 (SNS) and DINA at 100 nM, and EZH2i, EPZ-6438 (EPZ), and GSK343 (GSK) at 25 μM and 5 μM, respectively. Whole-cell lysates of TNBCs were immunoblotted with specific antibodies as indicated. All numbers under lanes indicated the relative amount of the images normalized with actin. h Ectopic expressing CDK2-phospho-mimetric EZH2 mutant abolishes CDK2i-induced ERα expression. The myc-EZH2T416D-expressing and vector control BT549 cells were treated with 50 nM DINA for 72 hours. Whole-cell lysates were immunoblotted with specific antibodies as indicated. T47D cell lysate was used for ERα control. Source data are provided as a Source Data file.
