Figure 5.

Docetaxel overcomes EZH2 inhibitor resistance in PTEN-mutated cancer cells in culture. (A, B) PTEN-positive 22Rv1 (A) and PTEN-negative C4-2 (B) cells were treated with different concentrations of GSK126 followed by MTS assay at different time points. (C) 22Rv1 and C4-2 cells were treated with different concentrations of GSK126 for 72 h and harvested for western blot analysis with the indicated antibodies. (D) C4-2 cells were treated with vehicle (DMSO) and GSK126 (10 μM) for 72 h followed by further treatment with or without DTX (2 nM) for 30 min prior to IFC. Cell membrane was stained with anti-E-cadherin; the nucleus was counterstained with DAPI. FNS stands for FOXO1 nuclear staining. Scale bar: 25 µm. (E) A hypothetical model deciphers repression of FOXO1 mRNA transcription by EZH2 and regulation of cellular localization of FOXO1 protein by taxane and the PI3K/PTEN/AKT pathway. “P” in a small red circle indicates phosphorylation. (F) C4-2 cells were treated with or without 10 μM of GS126 for 72 h and/or 2 nM of DTX for 30 min prior to fractionation assay and western blot analysis with indicated antibodies. Histone H3 and β-Tubulin were used as nuclear and cytosolic protein marker, respectively. (G - I) C4-2 cells were infected with lentivirus expressing nonspecific shRNAs (shNS) or FOXO1-specific shRNAs and selected with puromycin for stable cell lines. Cells were treated with or without 10 μM of GSK126 and/or 2 nM of DTX for 72 h and harvested for western blot analysis with indicated antibodies (G), MTS assay (H), and FACS analysis of percentage of sub G1 cells (I). Data are shown as means ± SEM. The P value was performed by the unpaired two-tailed Student's t-test. * P<0.05; ** P<0.01; *** P<0.001; n.s., no significance.