1. PERK signaling is constitutively activated upon EMT and promotes malignancy.
PERK signaling is constitutively activated upon EMT and promotes malignancy. A, Western blot analysis of EMT (shEcad and Twist) or control (shGFP) HMLE cells, luminal (MCF7, T47D, BT474, and ZR-75-30) and basal-B human breast cancer cell lines (SUM159, MDA-MB-231, MDA-MB-157, Hs578T, and BT549) for UPR pathway components; β-tubulin is used as a loading control. B, expression of GADD34 in EMT (shEcad and Twist) or control (shGFP) HMLE cells (left), luminal (MCF7, T47D, BT474, and ZR-75-3) and basal-B (SUM159, MDA-MB-231, MDA-MB-157, Hs578T, and BT549) human breast cancer lines (right). C, cell lysates from control (HMLE_shGFP) and EMT (HMLE_shEcad) cells treated with or without thapsigargin were collected. The lysates were then treated with or without lambda phosphatase, and PERK protein expression was analyzed by Western blotting. D, non-EMT (HMLE_shGFP) and EMT (HMLE_Twist) cells were treated with DMSO control, 40 nmol/L thapsigargin for 2 hours, or 1 μmol/L PERK inhibitor (PERKi) for 24 hours. Phosphorylated PERK (p-PERK) protein was analyzed by immunofluorescence staining. E, Western blotting for PERK and phospho-eIF2α expression in cell lysates from the Hs578T and SUM159 lines infected with control (shCtrl) or PERK-specific (shPERK) hairpins. F, non-EMT (HMLE_shGFP) and EMT (HMLE_shEcad and HMLE_Twist) cells were cotreated with 1.5 nmol/L thapsigargin and 0, 0.5 or 1 μmol/L of PERK inhibitor for 6 days. Surviving cells were quantified by cell counting. Data are represented as mean + SE from three replicates. G, HMLE_shGFP and HMLE_shEcad cells were pretreated with 1 μmol/L PERK inhibitor or vehicle control (DMSO) for 2 days before tumorsphere formation assay. The PERK inhibitor–pretreated cells were then cultured in tumorsphere-forming condition for another 4 days in the presence of 1 μmol/L PERK inhibitor, while the vehicle-pretreated cells were cultured in drug-free conditions for the same period of time. Representative bright-field images and quantification were shown. Scale bar, 50 μm. H, representative images of crystal violet staining of HMLE_shGFP and HMLE_shEcad cells pretreated with or without 1 μmol/L PERK inhibitor for 2 days following Transwell migration assay. Cells that migrated within 8 hours following seeding into 8-μm pore inserts were visualized and quantified. Scale bar, 50 μm. I, 4T1 cells were treated with DMSO control, 1 μmol/L PERK inhibitor for 3 days, or 2 nmol/L thapsigargin for 3 days followed by a 4-day recovery period in drug-free media. A total of 2 × 106 cells were injected into NOD/SCID mice via the tail vein, and lung tissues were harvested 15 days after injection. Representative images of mouse lung tissue stained with H&E are shown. Quantification of metastasis is also shown (5 mice per condition). *, P < 0.05; **, P < 0.01.
Yu-xiong Feng et al. Cancer Discovery 2014;4:
©2014 by American Association for Cancer Research