Fig. 8

AURKA-mediated phosphorylation of ALDH1A1 is crucial for ALDH1A1-induced EMT, CSC, and drug resistance. a ALDH1A1 expression increases CD44, Slug, and Snail promoter activities, whereas 3A-ALDH1A1 expression decreases CD44, Slug, and Snail promoter activities. BxPC3, ALDH1A1-BxPC3, and 3A-ALDH1A1-BxPC3 cells were plated in 96-well plates overnight and then transfected with CD44, Slug, Snail, and E-cadherin promoter-driven luciferase plasmids as described in Methods. After 48 h, luciferase activities were measured with the Dual Luciferase kit (Thermo Fisher Scientific). All experiments were done in triplicate, three independent times. *p < 0.05 and **p < 0.01, compared to control BxPC3 cells analyzed by two-way analysis of variance. b ALDH1A1 expression increases the levels of EMT and CSC markers while decreasing E-cadherin levels, whereas 3A-ALDH1A1 expression decreases the levels of EMT and CSC markers while increasing E-cadherin levels. c AURKA inhibition using MLN8237 decreases the levels of EMT and CSC markers, but increases E-cadherin levels. d ALDH1A1 overexpression increases sphere-forming ability in BxPC3 cells. BxPC3, ALDH1A1-BxPC3, and 3A-ALDH1A1-BxPC3 cells were incubated in ultralow attachment plates as described in Methods. Pancreatospheres were counted after 5 days. e ALDH1A1 overexpression increases drug resistance in BxPC3 cells. BxPC3, wild-type ALDH1A1-BxPC3, and 3A-ALDH1A1-BxPC3 cells were plated in 96-well plates overnight, doxorubicin (1 μM) was added, and cells cultured for another 24, 48, or 72 h. Cell viability was calculated by MTT assay. f AURKA ablation sensitizes BxPC3 cells to ALDH1A1 inhibition using N,N-diethylaminobenzaldehyde (DEAB). BxPC3 cells were either incubated with DEAB (100 μM) or with vehicle (DMSO). After 48 h, cell viability was analyzed. *p < 0.05 and **p < 0.01 compared to scrambled shRNA control by two-tailed Student’s t test. g ALDH1A1 inhibition sensitizes BxPC3 cells to AURKA inhibition. BxPC3 cells were treated with 100 μM DEAB and varying concentrations of MLN8237 (0–3 μM) for 24 h and cell viability analyzed. h Same data as in g but shown as line graph. i Proposed model showing AURKA-ALDH1A1 axis in promoting EMT, CSC, and chemoresistance in pancreatic cancer