Targeting breast cancer stem cells in triple-negative breast cancer using a combination of LBH589 and salinomycin

Abstract

The aim of this study is to investigate the efficacy of combining a histone deacetylase inhibitor (LBH589) and a breast cancer stem cells (BCSC)-targeting agent (salinomycin) as a novel combination therapy for triple-negative breast cancer (TNBC). We performed in vitro studies using the TNBC cell lines to examine the combined effect. We used the mammosphere and ALDEFLUOR assays to estimate BCSC self-renewal capacity and distribution of BCSCs, respectively. Synergistic analysis was performed using CalcuSyn software. For in vivo studies, aldehyde dehydrogenase 1 ALDH1-positive cells were injected into non-obese diabetic/severe combined immunodeficiency gamma (NSG) mice. After tumor formation, mice were treated with LBH589, salinomycin, or in combination. In a second mouse model, HCC1937 cells were first treated with each treatment and then injected into NSG mice. For mechanistic analysis, immunohistochemistry and Western blot analysis were performed using cell and tumor samples. HCC1937 cells displayed BCSC properties including self-renewal capacity, an ALDH1-positive cell population, and the ability to form tumors. Treatment of HCC1937 cells with LBH589 and salinomycin had a potent synergistic effect inhibiting TNBC cell proliferation, ALDH1-positive cells, and mammosphere growth. In xenograft mouse models treated with LBH589 and salinomycin, the drug combination effectively and synergistically inhibited tumor growth of ALDH1-positive cells. The drug combination exerted its effects by inducing apoptosis, arresting the cell cycle, and regulating epithelial-mesenchymal transition (EMT). Combination of LBH589 and salinomycin has a synergistic inhibitory effect on TNBC BCSCs by inducing apoptosis, arresting the cell cycle, and regulating EMT; with no apparent associated severe toxicity. This drug combination could therefore offer a new targeted therapeutic strategy for TNBC and warrants further clinical study in patients with TNBC.

Fig. 1

Breast cancer cell lines with BCSC properties. a Mammosphere formation assay in MCF7, SK-BR-3, MDA-MB-231, and HCC1937 cells. Images show representative mammospheres in each cell line at day 5 of incubation. Original magnification: ×200. b ALDEFLUOR assay in breast cancer cell lines (HCC1937, MDA-MB-231, MCF7, and SK-BR-3). The upper panels show the negative controls (cells treated with DEAB, a specific inhibitor of ALDH1). Based on the negative control, the gated area was assigned as ALDH1-positive. Percentages of ALDH1-positive cell populations of each cell lines are shown in the lower panels

Fig. 2

Effect of HDAC inhibitors on TNBC cell proliferation and mammosphere growth. HCC1937 and MDA-MB-231 cells treated with a LBH589 and b entinostat and cell viabilities assessed by MTT assay. c, d Single mammospheres collected and treated by DMSO, LBH589 (20 nM), and entinostat (300 nM). Pictures were taken on day 0, 2, 5, 7, 9, and 12. d Change in mammosphere volume ratio to day 0. The volume was calculated based on the radius of the mammosphere. Original magnification was ×200. The error bars represent mean ± SD. *p < 0.05; ***p <0.0005

Fig. 3

Effect of LBH589 and salinomycin on TNBC cell proliferation. a HCC1937 and MDA-MB-231 cells treated with parthenolide and salinomycin and cell viabilities assessed by MTT assay. b HCC1937 and MDA-MB-231 cells treated with a combination of parthenolide or salinomycin and LBH589 in different concentrations and cell viabilities assessed by MTT assay. c Synergistic analysis based on the MTT assay. The horizontal axis shows fraction affected (Fa) and the vertical axis shows combination index (CI). CI offers quantitative definition for additive effect (CI = 1), synergism (CI <1), and antagonism (CI > 1) in drug combinations

Fig. 4

Effect of LBH589 and salinomycin on TNBC BCSC self-renewal capacity and the TNBC BCSC population. a, b HCC1937 cells treated with DMSO, LBH589, salinomycin, or a combination of LBH589 and salinomycin in mammosphere culture conditions. a Images show representative mammospheres in each treatment at day 5. b Number of mammospheres>70 μm in each treatment group. c, d HCC1937 cells treated with DMSO, LBH589, salinomycin, or combination of LBH589 and salinomycin after ALDEFLUOR assays. c The panels show representative flow cytometry of ALDEFLUOR assays in each treatment condition. d The graph shows the ALDH1-positive cell population in each treatment condition. Original magnifications were ×200. The error bars represent mean ± SD. *p < 0.05; **p < 0.005; ***p < 0.005

Fig. 5

Effect of LBH589 and salinomycin on tumorigenicity of TNBC cells. a Tumor volume change in NSG mice. Mice were treated with vehicle, LBH589, salinomycin, or a combination of LBH589 and salinomycin. Error bars represent mean ± SEM. b Final weight (g) of harvested tumors. Images show the tumors in each treatment condition. Scale bar: 2 cm. The error bars represent mean ± SEM. *p < 0.05. (c, d) Body weight (g) and food intake (g) per mouse were measured weekly. Error bars represent mean ± SEM. e IHC staining of Ki67 from harvested formalin-fixed tumors in each treatment condition. The upper panel shows representative images of each treatment condition. The lower panel shows the percentage of Ki67-positive cells in each treatment condition. Original magnifications were ×400. Error bars represent mean ± SD. *p < 0.05; **p < 0.005; ***p < 0.005

Fig. 6

Effect of LBH589 and salinomycin on cell cycle arrest, apoptosis, and EMT-related markers in TNBC BCSCs. a HCC1937 cells treated with vehicle (DMSO), LBH589 (4, 16, 64 nM), salinomycin (SAL) (15 nM, 60 nM, 240 nM), or the drugs combined. After 5 days of treatment, cells were stained with 10 ug/ml of DAPI. Representative images from each treatment condition are shown. Original magnification was ×200. b HCC1937 cells treated with LBH589, SAL, or the drugs combined for 48 h. Cell extracts were analyzed by Western blotting with anti-cleaved PARP and anti-GAPDH antibodies. c HCC1937 cells treated with LBH589, SAL, or the drugs combined for 48 h. Cell extracts were analyzed by Western blotting with anti-acetylated histone H3 and H4, anti-p21, anti-cyclin D1, and anti-GAPDH antibodies. d HCC1937 ALDH1-positive and -negative cell extracts were analyzed by Western blotting with anti-E-cadherin, anti-vimentin, anti-N-cadherin, anti-SLUG, and anti-actin antibodies. e Proteins extracted from snap-frozen mouse tumors treated by vehicle, LBH589, SAL, or the drugs combined for 12 weeks and analyzed by Western blotting with anti-E-cadherin, anti-vimentin, and anti-actin antibodies