A class I histone deacetylase inhibitor, entinostat, enhances lapatinib efficacy in HER2-overexpressing breast cancer cells through FOXO3-mediated Bim1 expression

Abstract

Although there are effective HER2-targeted agents, novel combination strategies in HER2-overexpressing breast cancers are needed for patients whose tumors develop drug resistance. To develop new therapeutic strategy, we investigated the combinational effect of entinostat, an oral isoform-selective histone deacetylase type I inhibitor, and lapatinib, a HER2/EGFR dual tyrosine kinase inhibitor, in HER2+ breast cancer cells. We assessed the combinational synergistic effect and its mechanism by CellTiter Blue assay, flow cytometry, anchorage-independent growth, quantitative real-time PCR, small interfering RNA, Western blotting, and mammary fat pad xenograft mouse models. We found that compared with entinostat or lapatinib alone, the two drugs in combination synergistically inhibited proliferation (P < 0.001), reduced in vitro colony formation (P < 0.05), and resulted in significant in vivo tumor shrinkage or growth inhibition in two xenograft mouse models (BT474 and SUM190, P < 0.001). The synergistic anti-tumor activity of the entinostat/lapatinib combination was due to downregulation of phosphorylated Akt, which activated transcriptional activity of FOXO3, resulting in induction of Bim1 (a BH3 domain-containing pro-apoptotic protein). Furthermore, entinostat sensitized trastuzumab/lapatinib-resistance-HER2-overexpressing cells to the trastuzumab/lapatinib combination and enhanced the anti-proliferation effect compare with single or double combination treatment. This study provides evidence that entinostat has enhanced anti-tumor effect in combination with HER2-targeted reagent, lapatinib, and resulting in induction of apoptosis by FOXO3-mediated Bim1 expression. Our finding justifies for conducting a clinical trial of combinational treatment with entinostat, lapatinib, and trastuzumab in patients with HER2-overexpressing breast cancer resistant to trastuzumab-based treatment.

Fig. 1

The combination of entinostat and lapatinib inhibited proliferation of HER2+ breast cancer cells. a Cells (2×10⁵) were placed on a 6-well plate overnight, treated with or without the drugs (entinostat, 5.0 μM for all cell lines; lapatinib, 1.0 μM for HER2+ cell lines and 5.0 μM for HER2- cell lines) for 72 hours, and then stained with propidium iodide (PI) for cell cycle analysis using flow cytometry. Each bar represents the mean of 3 independent experiments; error bars, SD. *** p <0.001 combination compared with either entinostat or lapatinib. b Cells were treated with entinostat or lapatinib or both in combination for 72 hours. For combination, entinostat was fixed with 0.5 μM and mixed with various range of lapatinib (0.01 – 20 μM). And then a WST-1 proliferation assay was performed. For analysis, the non-linear fit curve method was used via GraphPad Prism software. The table represents the synergistic inhibitory effect of entinostat and lapatinib. The fractional inhibitory concentration (FIC) for the combination is the sum of the FICs of the 2 drugs and was interpreted as follows: < 0.5, synergy; 0.5 – 2.0, additive; > 2.0, antagonistic.

Fig. 2

Combined entinostat and lapatinib at clinically relevant doses inhibited the cell cycle and induced apoptosis. a Cells (2×10⁵) were placed on a 6-well plate overnight and then treated with or without the drugs (entinostat, 1.0 μM for all cell lines; lapatinib, 1.0 μM for SUM190 and 0.1 μM for BT474) for 48 hours. After the cells were collected, a PI staining assay was performed for cell-cycle analysis using flow cytometry. b Cells (2×10⁵) were placed on a 6-well plate overnight, and then treated with or without the drugs (entinostat, 1.0 μM for all cell lines; lapatinib, 1.0 μM for SUM190 and 0.1 μM for BT474) for 48 hours. An Annexin V/7AAD staining assay was performed for detection of apoptosis using flow cytometry. * p <0.05 combination compared with lapatinib. ** p <0.01 combination compared with either control or entinostat. Data shown are representative of 3 experiments with similar results.

Fig. 3

The combination of entinostat and lapatinib enhanced drug efficacy in terms of in vitro colony formation in a soft agar assay. For visualizing or counting, colonies were stained with 200 μL of MTT (1 mg/mL) solution for 2 hours and counted using the GelCount colony-counting system according to the manufacturer's instructions. Statistical significance was evaluated by t test using GraphPad Prism software. a BT474. b SUM190. Cells (2×10³) were seeded into soft agar with the indicated drug(s) and incubated for 3 weeks. Scale bars, 100 μm. Bars in the graph represent means; error bars, standard deviation. * p <0.05 combination compared with entinostat. ** p <0.01 combination compared with either control or lapatinib. Data shown are representative of 3 experiments with similar results.

Fig. 4

The combination of entinostat and lapatinib effectively suppressed tumor growth in breast cancer xenograft model. Cells (1×10⁷ for BT474 or 2×10⁶ for SUM190) in 50% Matrigel solution were transplanted into the mammary fat pads of 8 female nude mice per group. a BT474. b SUM190. Treatment started on day 18 after tumor cell implantation. The administered volume was 0.2 ml/30 g mouse body weight. Treatment was continued for 70 days for the BT474 group, and 25 days for the SUM190 group. IHC staining represents quantification of KI-67-positive cells in tumor tissue samples from each treatment group. Graphs and statistical significance were evaluated by an ANOVA using GraphPad Prism software. Error bars, standard deviation; magnification, ** p <0.01, *** p <0.001, ×20; scale bar, 50 μm.

Fig. 5

The enhanced anti-tumor effect of the entinostat/lapatinib combination depends on FOXO3a-mediated Bim1 expression. a Combination treatment or single-agent lapatinib treatment inhibited pFOXO3a phosphorylation at the Ser253 position. Cells were treated with drugs for 24 hours, and then total cell lysates were collected for Western blotting. The ratios of pFOXO3a-S253are shown above the blots. b Cytosol-nucleus fractionation. Cells (2×10⁶) were placed on a 6-well plate overnight and then treated with or without the drugs for 24 hours. U1 snRNP 70, a measure of FOXO3a, was assessed by Western blotting. The ratios of FOXO3a are shown above the blots. c Quantitative RT-PCR. Cells (2×10⁵) were placed on the 6-well plate overnight and then treated with or without the drugs (entinostat, 1.0 μM for both cell lines; lapatinib, 0.1 μM for BT474 and 1.0 μM for SUM190) for 24 hours. Equal amounts of total RNA (20 ng for each sample) were mixed, and target genes were amplified with a specific primer set. ** p <0.01 compared combination with either control or single treatment. d Western blot assay. Cells (2×10⁵) were placed on a 6-well plate overnight, and then treated with or without the drugs for 48 hours. e Effect of siFOXO3 on Bim1 expression. Cells were pre-treated with scrambled siRNA or siFOXO3a for 48 hours, followed by entinostat/lapatinib treatment for 48 hours. Cell lysates were harvested and Western blotting performed. The ratios of Bim1are shown above the blots. f, g Depletion of Bim1 by siBim1 protected entinostat/lapatinib-induced cell death in BT474 and SUM190. Cells were pre-treated with scrambled siRNA or siBim1 for 48 hours, followed by entinostat/lapatinib treatment for 72 hours. Cell viability was assessed using a WST-1 assay, *** p <0.001 compared siControl with siBim1.

Fig. 6

Immunohistochemical staining and quantification of Bim1 expression in representative tumor samples from each treatment group. a BT474. b SUM190 with low-dose treatment. c SUM190 with high-dose treatment. The images were converted by ImageJ software to accomplish quantification of Bim1 expression. Data shown are representative of 3 IHC staining experiments with similar results. Magnification, ×20; scale bar, 50 μm. Bars in graph represent means; error bars, standard deviation. ** p <0.01, *** p <0.001 compared combination with either control or single treatment.

Fig. 7

Entinostat enhances trastuzumab/lapatinib efficacy in trastuzumab/lapatinib-resistant HER2+ breast cancer cells. Trastuzumab/lapatinib-resistant SUM190 cells (SUM190-TLR) were obtained by continuously exposing SUM190 cells (which are known to be trastuzumab resistant) to lapatinib (5 μg/ml) for 6 months. Dose-effect curve was analyzed using CalcuSyn software (Biosoft, Cambridge, UK). Combination index (CI) of entinostat, lapatinib and trastuzumab in SUM190-TLR cells: A quantitative measure of the degree of drug interaction. CI value: <0.1 indicates very strong synergism; 0.10-0.30, strong synergism; 0.30-0.70, synergism; 0.70-0.85, moderate synergism; 0.85-0.90, slight synergism; 0.90-1.10, nearly additive; 1.10-1.20, slight antagonism; 1.20-1.45, moderate antagonism; 1.45-3.3, antagonism; 3.3-10, strong antagonism; >10, very strong antagonism. Fractional index (Fa), the fraction of cells affected by the dose.