Kinase Inhibitors Screen Tamoxifen Resistant Breast Cancer
Antiestrogen resistance of breast cancer has been related to enhanced growth factor receptor expression and activation. We have previously shown that ectopic expression and subsequent activation of the insulin-like growth factor-1 receptor (IGF1R) or the epidermal growth factor receptor (EGFR) in MCF7 or T47D breast cancer cells results in antiestrogen resistance. In order to identify critical therapeutic targets that mediate this antiestrogen resistance, we performed a kinase inhibitor screen with 273 different inhibitors. Various ALK inhibitors, including TEA684, AZD3463 and LDK378, inhibited cell proliferation in IGF1R expressing cells under both normal and antiestrogen resistance conditions by preventing IGF1R activation and subsequent downstream signaling; ALK inhibitors did not affect EGFR signaling. On the other hand, MEK1/2 inhibitors, including PD0325901, selumetinib, trametinib and TAK733, selectively antagonized IGF1R signaling-mediated antiestrogen resistance but did not affect cell proliferation under normal growth conditions. The MEK inhibitors PD0325901, selumetinib, trametinib and TAK733 caused an arrest in the G0/G1 cell cycle phase only under antiestrogen resistance conditions in IGF1R expressing cells, but not under normal growth conditions. RNAseq analysis revealed that MEK inhibitors PD0325901 and selumetinib drastically altered cell cycle progression and cell migration networks under IGF1R signaling-mediated antiestrogen resistance. In a group of 291 patients with primary operable ER+ breast cancer, strong pMEK staining in metastatic breast cancer after first-line tamoxifen treatment was significantly related to no clinical benefit. We propose a critical role for MEK activation in IGF1R signaling-mediated antiestrogen resistance and anticipate that dual-targeted therapy with MEK inhibitor and antiestrogen could improve treatment outcome.
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Parental MCF7 (MCF7/WT) cells and MCF7/EGFR and MCF7/IGF1R cells were cultured in RPMI 1640 L-Glutamine 25mM HEPES (Gibco, via Thermo Fisher Scientific; Waltham, MA, USA), supplemented with 10 % v/v fetal bovine serum (FBS) (Gibco, Life Technologies; Grand Island, NY, USA) and penicillin/ streptomycin (125 Units/mL and 125 µg/mL) (Invitrogen; Grand Island, NY, USA) (RPMI++ medium). Parental T47D (T47D/WT) cells (American Type Culture Collection; Manassas, VA, USA) and T47D/IGF1R cells were cultured in DMEM with D-glucose, L-Glutamine and pyruvate (Gibco, Life Technologies; Grand Island, NY, USA), supplemented with 10 v/v % FBS and penicillin/ streptomycin (125 Units/mL and 125 µg/mL)(DMEM++ medium). All cells were cultured at 37˚C and 5 % carbon dioxide. For hormone and growth factor starvation, cells were maintained for 48 h in starvation medium consisting of phenol red free RPMI 1640x medium (Gibco, Life Technologies; Grand Island, NY, USA) supplemented with 5 % v/v charcoal dextran-treated fetal bovine serum (CDFBS) (Hyclone, Thermo Scientific; Waltham, MA, USA) and penicillin/streptomycin. Proliferation assay. All cells were plated at a density of 10,000 cells/well in 96-well plates (Corning; NY, USA), allowed to attach overnight and thereafter maintained in starvation medium for 48 hours. Subsequently, estrogen (E2), growth factor (EGF or IGF1), and compound (4OHT and kinase inhibitor) were added, and cells were allowed to proliferate for another 96 hours. Tamoxifen resistant (TamRes) culturing condition for MCF7/IGF1R and T47D/IGF1R was phenol red free RPMI 1640x medium with 5% v/v CDFBS, 1 μM 4OHT, 1 nM E2 and 100 ng/ml IGF1 [12,30]. TamRes culturing condition for MCF7/EGFR was phenol red free RPMI 1640x medium with 5% v/v CDFBS, 0.1 μM 4OHT, 0.1 nM E2 and 100 ng/ml EGF . After 96 h cell number was determined by a colorimetric SRB assay (39). This assay was previously adapted and validated by us for the cells used in this study (11, 12). In short, cells were fixed with 20 µl 50 % w/v TCA in 1 v/v % acetic acid for 1 hour at 4°C, washed five times with tap water, and air-dried. Thereafter cells were stained with 0.4 % w/v SRB in 1 % v/v acetic acid at room temperature for 30 min, washed five times in 1 % v/v acetic acid and air-dried overnight. Subsequently, protein-bound SRB in the wells was dissolved in 200 µl 10 mM unbuffered Tris solution (pH>10) and absorbance was measured at 540nm in a plate reader. The kinase inhibitor screen was performed with 273 kinases targeting 42 cancer-related kinases. All inhibitors were tested in duplicate at a concentration of 1 μM alongside different concentrations of BMS-536924 (for MCF7/IGF1R cells) or Lapatinib (for MCF7/EGFR cells), and DMSO and 4-OHT only. Data from the inhibitor screen was analyzed by unbiased sample-based analysis in which SRB absorbance values of all individual wells samples were converted to z-scores