The human breast cancer cell lines, MDA-MB-435, MDA-MB-231, MDA-MB-468, SKBR3 and MDA-MB-453, were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI-1640 or DMEM supplemented with 10% fetal bovine serum at 37°C with 5% CO₂ in a humidified incubator.

Lapatinib was kindly provided by GlaxoSmithKline. AZD6244 was purchased from Selleck Chemicals (Woburn, MA). LY294002 was purchased from Cell Signaling Technology (Beverly, MA). In these cases, 10 mmol/l aliquots of drug in DMSO (dimethyl sulfoxide) were stored at-20°C and diluted just before utilization. All antibodies were purchased from commercial sources as indicated below: anti-Neu (F-11), anti-p-Neu (Tyr1248)-R, anti-ErbB-3 (C-17), anti-p-ErbB-3 (Tyr1328), anti-p-Akt1/2/3 (Ser 473)-R, anti-p-Erk (E-4), anti-Akt (Ser 473)-R, anti-Erk (E-4), NF-κB p50 and β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). EGFR, p-EGFR, phospho-NF-κB p65 (Ser536), NF-κB p65 (C22B4), IκB-α (L35A5), phospho-IκB-α (Ser32) and histone H3 antibodies were obtained from Cell Signaling Technology. HRP-conjugated goat-anti-rabbit IgG, goat-anti-mouse IgG and donkey-anti-goat IgG antibodies were purchased from Santa Cruz Biotechnology, Inc. or Cell Signaling Technology.

To generate lapatinib-resistant SKBR3 cells (rSKBR3), parental SKBR3 cells were initially grown in medium containing 0.25 μmol/l lapatinib. The concentration of lapatinib in medium was gradually increased to 5 μmol/l over 18 months. The resistant cells were constantly pooled by collecting all viable cells of several plates onto 1 plate. Single-cell clones were isolated from the pooled resistant cells. Finally, the resistant cells were maintained in medium supplemented with 5 μmol/l lapatinib. Resistance to lapatinib was confirmed by a cell viability assay as described below. The resistant cells were cultured in medium without lapatinib for 2 or 3 days before each experiment.

Cell viability was determined using the CCK-8 (Cell Counting Kit-8; Dojindo, Japan) assay. Briefly, parental and resistant cells were seeded at a density of 3–5×10³ cells into a 96-well microplate and incubated overnight. The cells were then treated with various concentrations of lapatinib. After incubation for 48 h, 10 μl of CCK-8 solution was added to each well, and the plates were further incubated for 3 h at 37°C. The absorbance at 450 nm was measured with an absorbance microplate reader. Cell survival for all experiments was expressed as the percentage of viable cells compared with untreated cells.

Cells were washed twice in cold PBS and then lysed in RIPA buffer [150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 0.25% (w/v) deoxycholate, 1% NP-40, 5 mM sodium orthovanadate, 2 mM sodium fluoride and a protease inhibitor cocktail]. The protein concentration of the supernatants was determined using a modification of the Bradford method. Equal amounts of proteins (40 μg) were resolved by SDS-PAGE. Proteins were transferred to PVDF membranes and blocked with 5% non-fat milk in PBS-T. Membranes were incubated overnight with specific antibodies. After four washes in PBS-T, membranes were then incubated with horseradish peroxidase-linked secondary antibody at a 1:2,000 dilution. Target proteins were visualized with the SuperSignal West Femto Maximum Sensitivity Substrate kit and subsequent exposure to X-OMAT X-ray film according to the manufacturer’s instructions.

Nuclear extracts were prepared using a nuclear and cytoplasmic proteins extract kit purchased from Beyotime Institute of Biotechnology (Haimen, China). Electrophoretic mobility shift assay (EMSA) was performed using a chemiluminescent assay kit according to the protocol provided by the vendor (Beyotime Institute of Biotechnology) with minor modifications. Briefly, for each reaction, 20 μg of nuclear protein extracts was incubated with biotin end-labeled probe. The protein-DNA complex was then resolved by electrophoresis on 5% native polyacrylamide gel and transferred to a nylon membrane. DNA on the membrane was immediately cross-linked for 10 min using an UV cross-linker. The biotin end-labeled DNA probe was detected using streptavidin conjugated to horseradish peroxidase (HRP) and a chemiluminescent substrate. Biotin-labeled double-strand NF-κB consensus oligonucleotide (5′-AGT TGA GGG GAC TTT CCC AGG C-3′) and Oct-1 oligonucleotide (5′-TGT CGA ATG CAA ATC ACT AGA A-3′) were obtained from Beyotime Institute of Biotechnology.

To determine the effect of lapatinb on the activity of NF-κB, we performed EMSA. In SKBR3 cells, lapatinib inhibited activation of NF-κB in a dose-dependent manner (Fig. 1). Similarly, a high dose of lapatinib (5 μmol/l) markedly reduced the activity of NF-κB in another HER2-overexpressing breast cancer cell line, MDA-MB-453. In contrast, lapatinib had almost no impact on the activity of NF-κB in non-HER2-overexpressing breast cancer cell lines, including MDA-MB-231, MDA-MB-468 and MDA-MB-435 (Fig. 1). These results suggest that lapatinib inhibits the activation of NF-κB in HER2-overexpressing breast cancer cells.

To better understand how lapatinib inactivates NF-κB in HER2-positive breast cancer cells, we performed western blot analysis. As expected, lapatinib reduced the expression levels of p-HER2, p-Akt and p-Erk proteins in a dose-dependent manner in SKBR3 cells (Fig. 2A). Importantly, treatment with lapatinib resulted in a decreased phosphorylation of IκB-α in SKBR3 cells, whereas lapatinib had almost no impact on the expression levels of total HER2, Akt, Erk, p65 and p-p65 proteins (Fig. 2A). We then treated SKBR3 cells with 2 μmol/l lapatinib for different times, and measured the protein expression levels of HER2 and NF-κB pathways by western blot analysis. As shown in Fig. 2B, lapatinib reduced the phosphorylation of IκB-α in a time-dependent manner, whereas lapatinib increased the expression level of total IκB-α protein. To further confirm the inhibitory role of lapatinib on NF-κB activity, we evaluated cellular localization of p65 by western blot analysis. Interestingly, lapatinib inhibited p65 nuclear accumulation in a dose-dependent manner in SKBR3 cells (Fig. 2C).

To measure the relative resistance of parental SKBR3 and rSKBR3 cells to lapatinib, we performed CCK-8 assays. As indicated in Fig. 3A, the 50% inhibitory concentrations (IC₅₀) of lapatinib in parental SKBR3 cells, pooled resistant cells and representative clone were ~0.3, 3.7 and 3.8 μmol/l, respectively, suggesting that SKBR3 cells gradually became insensitive to lapatinib. We next assayed the expression levels of several HER signaling pathway proteins in the parental and resistant rSKBR3 cells by immunoblot analysis. The expression levels of p-HER2, p-HER3, p-EGFR, p-Erk and p-Akt proteins decreased in rSKBR3 cells in comparison with parental SKBR3 cells, while the expression levels of total HER2, HER3, EGFR, Erk and Akt proteins had almost no change (Fig. 3B). This result demonstrated that lapatinib still retained its inhibitory role of EGFR/HER2 signaling in our resistant cell model.

We next examined whether the activity of NF-κB changed in our cell model. A moderately decreased NF-κB activity was observed in rSKBR3 cells, compared with parental SKBR3 cells (Fig. 3C).

We next assayed the expression levels of NF-κB pathway proteins by western blot analysis. As showed in Fig. 3D, the expression level of the p-IκB-α protein was markedly decreased in the pooled resistant cells and representative clone, when compared with the parental SKBR3 cells. The expression levels of p-p65, p65 and p50 had no significant difference between the parental and resistant rSKBR3 cells (Fig. 3D). We further evaluated the cellular localization of p65 by western blot analysis. Nuclear accumulation of p65 was decreased in the resistant cells, compared with the parental SKBR3 cells (Fig. 3E).

We previously showed that lapatinib inhibits activation of the PI3K/Akt and Erk pathways in SKBR3 cells (6). Thus it was tempting to speculate that lapatinib may inhibit NF-κB activation through blocking these pathways. To address this possibility, we treated SKBR3 and MDA-MB-453 cells with the PI3K inhibitor LY294002 and the MEK inhibitor AZD6244, and measured the activity of NF-κB by EMSA. As indicated in Fig. 4A, a deceased NF-κB activity was observed in the SKBR3 cells treated with LY294002. In MDA-MB-453 cells, LY294002 inhibited activation of NF-κB in a dose-dependent manner (Fig. 4C). However, AZD6244 had relatively little impact on NF-κB DNA binding in SKBR3 cells (Fig. 4B), as well as in MDA-MB-453 cells (Fig. 4D).

We next examined the impact of LY294002 and AZD6244 on the expression level of NF-κB pathway proteins by western blot analysis. In SKBR3 cells, LY294002 reduced phosphorylation of Akt in a concentration-dependent manner (Fig. 4E). Importantly, phosphorylation of IκB-α was potently inhibited by LY294002, while the expression level of total IκB-α was increased, particularly in the presence of the highest dose of LY294002 (Fig. 4E). As expected, phosphorylation of Erk was reduced by AZD6244 in a dose-dependent manner in SKBR3 cells, whereas there was no detectable change in IκB-α phosphorylation in SKBR3 cells (Fig. 4F). AZD6244 had almost no effect on the expression levels of p-p65, p65, p50 and total IκB-α.