Figure 3
Endogenous KLF4/5 mediate lapatinib resistance in breast cancer models. (a) Levels of KLF4/5 in primary human breast tumors were determined by RNAseq (Illumina HiSeq RNAseqV2). Upper quartile normalized data were downloaded from TCGA and assigned a PAM50 subtype. Spearman’s correlation was performed on the log2 transformed data. (b) BT474 cells were treated with DMSO or lapatinib for the indicated interval. Whole-cell lysate was analyzed by western blot. Expression levels from three independent experiments were determined using ImageJ for quantitation, with normalization to β-actin (bars, S.D.). (c) BT474 cells were treated with trastuzumab or sterile water for the indicated interval and whole-cell lysate was analyzed by western blot. (d) KLF4/5 transcript levels were determined by qRT-PCR following lapatinib exposure. Expression data were normalized using the housekeeping gene B2M. (e) The pMIR-Report-Luc-KLF4-FL translation reporter contains as an insert within the FLuc 3’ UTR the full-length KLF4 transcript, including the KLF4 protein coding region and the flanking UTRs, as previously described.18 Translation efficiency was measured by determining normalized Fluc activity in BT474 cells treated for 24 h with lapatinib or DMSO (left panel). miR-206 levels were determined by qRT-PCR following 24-h lapatinib exposure. Expression data were normalized using U6 snRNA (right panel). (f) Cells were treated with the indicated shRNA construct, and the resulting cell populations were treated with lapatinib for 96 h. For each cell population, cell viability relative to the DMSO control was obtained via ATP-based luminescence assay (bars, S.D.). (g) Similarly, the lapatinib effect on the relative cell viability of M6 cells expressing ectopic KLF4 and/or KLF5 was determined. Empty vector served as a control. (h) To assess MMI, M6 cells were treated with lapatinib for 24 h, stained with 250 nM of Mitotracker dye and analyzed by flow cytometry. (i) To assess activity of the intrinsic apoptotic pathway, caspase-9 levels were determined in M6 cells expressing shCtl, shKLF4, shKLF5 or shKLF4/5. Cells were treated with lapatinib for 24 h before preparation of cell extracts. *P<0.05; **P<0.01; ***P<0.001
