Fig. 7
Downregulation of the extracellular signal-related protein kinase (ERK) pathway is critical for the conversion of breast cancer cells derived from chemotherapy-treated patients (CT-PCs) into CT + shear stress (SS) displaying cancer stem-like cell (CSLC)/tumor-initiating cell (TIC) properties. a Transcriptional profile of the selected ERK-related genes (Elk1, Ets1, Mcl1, Tp53, and Stat3). b Western blot analysis of p-ERK, ERK, p-p38, p38, p-JNK, JNK, and p53 from CT-PCs or CT + SS (10 days) n. c CT-PCs under +SS for 3 days were treated with PD98059 (mitogen-activated protein kinase (MEK) inhibitor), SB203580 (p38 MAPK inhibitor), and SP600125 (JNK inhibitor) for 24 h and subjected to sphere-formation assay (*p < 0.01 compared with dimethyl sulfoxide (DMSO)-treated cells). Error bars represent ± SD from the three independent experiments. d CT + SS obtained from 3-day culture under +SS were transfected with Flag-tagged wild-type MEK (WT-MEK) or active MEK (active-MEK), and the level of Flag, p-MEK, MEK, p-ERK, ERK, p53, and p21 were assessed by western blot. e Changes in the number of sphere-forming cells in WT-MEK or active-MEK-expressing CT-PCs are shown as percent changes (%) (*p < 0.01, compared to mock transfection). f Relative changes in the expression of self-renewal marker (Nanog, Oct4B, and Sox2) and multi-drug resistance (Aldh1, Abcg2, and Abcb5) genes are normalized to Gapdh. Data represent mean ± SD from three independent experiments (*p < 0.01). g In vivo tumorigenicity of mock, WT-MEK, and active-MEK carrying CT + SS implanted subcutaneously on NOD/SCID mice. Tumor volumes and weights were measured in NOD/SCID mice after inoculation with 2 × 105 cells for 4 weeks. h Western blot analysis of p-GSK3β and GSK3β from CT-PCs or suspension CT + SS are shown. CT-PCs under +SS for 3 days were treated with PD98059 (MEK inhibitor) or/and BIO (GSK3β inhibitor) for 24 h and subjected to sphere-formation assay (i), quantitative real-time RT-PCR (j and upper panels of k and l), or western blot (lower panels of k and l) analyses of stemness (Nanog, Oct4B, and Sox2; j, EMT (Snail, Twist, and N-Cadherin (k)), and epithelial (E-Cadherin, Claudin-7, and Cytokeratin-8; l) marker genes. Relative changes in the expression of stemness and EMT marker genes are normalized to Gapdh; *p < 0.01, Error bars correspond to mean ± SD