Niclosamide sensitizes triple-negative breast cancer cells to ionizing radiation in association with the inhibition of Wnt/β-catenin signaling

Abstract

Triple-negative breast cancer (TNBC) is one of the most difficult breast cancers to treat because there is no targeted treatment, and conventional cytotoxic chemotherapy followed by adjuvant radiation therapy is the standard of care for patients with TNBC. We herein reported that ionizing radiation (IR) induced Wnt3a, LRP6 and β-catenin expression and consequently activated Wnt/β-catenin signaling in TNBC MDA-MB-231, MDA-MB-468 and Hs578T cells. Moreover, depletion of β-catenin by shRNA sensitized TNBC cells to IR, whereas treatment of Wnt3a protein or overexpression of β-catenin resulted in radioresistance of TNBC cells. Niclosamide, a potent inhibitor of Wnt/β-catenin signaling, not only inhibited constitutive Wnt/β-catenin signaling, but also blocked IR-induced Wnt/β-catenin signaling in TNBC cells. In addition, niclosamide sensitized TNBC cells to IR, prevented Wnt3a-induced radioresistance, and overcame β-catenin-induced radioresistance in TNBC cells. Importantly, animals treated with the combination of niclosamide and γ-ray local tumor irradiation had significant inhibition of MDA-MB-231 tumor growth compared with treated with local tumor irradiation alone. These findings indicate that Wnt/β-catenin signaling pathway plays an important role in the development of radioresistance of TNBC cells, and that niclosamide had significant radiosensitizing effects by inhibiting Wnt/β-catenin signaling in TNBC cells. Our study also provides rationale for further preclinical and clinical evaluation of niclosamide in TNBC management.

Keywords: Wnt/β-catenin signaling; niclosamide; radiosensitization; triple-negative breast cancer.

Figure 1. IR induces activation of Wnt/β-catenin signaling in TNBC cells

A. MDA-MB-231, MDA-MB-468 and Hs578T cells were irradiated with indicated doses of γ-ray IR. After 6 h incubation, levels of Wnt3a, p-LRP6 (S1490), LRP6, p-β-catenin (S675), β-catenin, C-myc, survivin were analyzed by Western blotting. All the samples were also probed with anti-β-actin and anti-vinculin antibody to verify equal loading. B. The bands of each protein in above experiments were quantified by densitometric analysis, and normalized to the corresponding level of β-actin. C. MDA-MB-231, MDA-MB-468 and Hs578T cells were irradiated with indicated doses of γ-ray IR. After 6 h incubation, the mRNA levels of C-myc and survivin were examined by real-time RT-PCR. Values are averages of three independent experiments with the standard deviations indicated by error bar. ^*P<0.05, ^**P<0.01, ^***P<0.001 versus corresponding non-irradiated cells.

Figure 2. Niclosamide inhibits Wnt/β-catenin signaling in TNBC cells

A, B. MDA-MB-231, MDA-MB-468 and Hs578T cells were treated with γ-ray IR (6 Gy) in the absence or presence of niclosamide (1.5 μM) for 24 h. Levels of Wnt3a, p-LRP6 (S1490), LRP6, p-β-catenin (S675), β-catenin, C-myc and survivin were analyzed by Western blotting. All the samples were also probed with anti-β-actin antibody to verify equal loading. The bands of each protein were quantified by densitometric analysis, and normalized to the corresponding level of β-actin. Values are averages of three independent experiments with the standard deviations indicated by error bar. C, D. MDA-MB-231 and MDA-MB-468 cells were treated with the same conditions as in (A). The cells were fixed, permeabilized and immunolabeled for β-catenin. β-catenin nuclear staining was detected by fluorescent microscopy (1000×magnification; Scale bar, 10 μm). The relative mean fluorescence intensity of nuclear β-catenin staining was calculated out of a total number of 200 cells per sample. Values are averages of three independent experiments with the standard deviations indicated by error bars. ^*P<0.05, ^**P<0.01, ^***P<0.001 versus corresponding cells treated with DMSO control; ^$$p<0.01, ^$$$p<0.001 versus corresponding cells treated with IR alone. Niclo, niclosamide.

Figure 3. Niclosamide sensitizes TNBC cells to IR

A, B. MDA-MB-231 cells were treated with γ-ray IR (6 Gy) and MDA-MB-468 cells were treated with X-ray IR (8 Gy) in the absence or presence of niclosamide (1.5 μM) for 24 h. Levels of apoptosis were examined by TUNEL staining at 48 h post-irradiation (400× magnification; Scale bar, 10 μm). Values are averages of three independent experiments with the standard deviations indicated by error bars. ^***P<0.001 versus corresponding cells treated with DMSO control; ^$p<0.05 versus corresponding cells treated with IR alone. C. MDA-MB-231, MDA-MB-468 and Hs578T cells were seeded into 60-mm dishes in duplicate, and were treated with γ-ray IR at indicated doses in the absence or presence of niclosamide at indicated concentrations for 24 h. After incubation for 10 to 14 days, the colonies with more than 50 cells were counted. The plating efficiency (PE) and the sensitizer enhancement ratio (SER) were determined as described in MATERIALS AND METHODS. Values are averages of three independent experiments with the standard deviations indicated by error bars. ^*P<0.05, ^**P<0.01, ^***P<0.001 versus corresponding control cells. Niclo, niclosamide.

Figure 4. Niclosamide suppresses β-catenin-induced radioresistance in TNBC cells

A. MDA-MB-231 cells were transfected with β-catenin shRNA or control vector. After 48 h incubation, the levels of β-catenin were examined by Western blotting. B. MDA-MB-231 cells in 60-mm dishes in duplicate were transfected with control vector, β-catenin shRNA, or control vector with niclosamide treatment, and treated with γ-ray IR at indicated doses. The colonies with more than 50 cells were counted after 12 to 14 days. C. MDA-MB-231 cells were transfected with β-catenin cDNA or vector. After 48 h incubation, the levels of β-catenin were examined by Western blotting. D. MDA-MB-231 cells in 60-mm dishes in duplicate were transfected with β-catenin cDNA or vector, and treated with γ-ray IR at indicated doses in the absence or presence of niclosamide (1.5 μM) for 24 h. After incubation for 10 to 14 days, the colonies with more than 50 cells were counted. PE and SER were determined as described in MATERIALS AND METHODS. Values are averages of three independent experiments with the standard deviations indicated by error bars. For SF curve, ^*P<0.05, ^**P<0.01, ^***P<0.001 versus corresponding cells treated with DMSO control. For SER value, ^**P<0.01, ^***P<0.001 versus corresponding cells transfected with control vector and treated with DMSO; ^###P<0.001 versus corresponding cells transfected with β-catenin plasmid. Niclo, niclosamide.

Figure 5. Niclosamide suppresses Wnt3a-induced Wnt/β-catenin signaling in TNBC cells and prevents Wnt3a-induced radioresistance

A. MDA-MB-231 cells were seeded into 60-mm dishes in triplicate, and were treated with X-ray IR at indicated doses in the absence or presence of niclosamide (1.5 μM) for 24 h and Wnt3a (50 ng/ml) for 8 h. After incubation for 10 to 14 days, the colonies with more than 50 cells were counted. PE and SER were determined as described in MATERIALS AND METHODS. Values are averages of three independent experiments with the standard deviations indicated by error bars. ^**P<0.01, ^***P<0.001 versus corresponding cells treated with DMSO control. ^###P<0.001 versus corresponding cells treated with Wnt3a alone. B. MDA-MB-231 cells were seeded into 60-mm dishes, and were treated with X-ray IR (8Gy) in the absence or presence of niclosamide (1.5 μM) for 24 h and Wnt3a (50 ng/ml) for 8 h. Levels of Wnt3a, p-LRP6 (S1490), LRP6, p-β-catenin (S675), β-catenin, C-myc and survivin were analyzed by Western blotting. All the samples were also probed with anti-β-actin antibody to verify equal loading. Niclo, niclosamide.

Figure 6. In vivo antitumor efficacy of niclosamide and IR in the TNBC MDA-MB-231 xenograft mouse model

A. The treatment schedule of niclosamide and IR. B. Growth curve of tumor of nude mice treated with vehicle (DMSO:Tween 80:H₂O= 3:4:8), niclosamide (20 mg/kg/dose), IR (10 Gy γ-ray at day 7), and niclosamide plus IR as described in MATERIALS AND METHODS. C. Nude mice were sacrificed and the tumors excised and weighed at the end of experiments. D. Body weights of nude mice treated with vehicle, niclosamide, IR, and niclosamide plus IR at the dosage and schedule as described in (B). E. Immunohistochemical staining with anti-β-catenin antibody of MDA-MB-231 tumors (400×magnification; Scale bar, 25 μm). F. Immunohistochemical staining with anti-Ki67 antibody of MDA-MB-231 tumors (400×magnification; Scale bar, 25 μm). G. Levels of cleaved caspase-3 in MDA-MB-231 tumors were examined by Western blotting. Data shown in (B-F) are averages from five nude mice. ^*P<0.05, ^**P<0.01, ^***P<0.001 versus nude mice treated with vehicle control; ^#p<0.05, ^##p<0.01, ^###p<0.001 versus nude mice treated with IR alone. Niclo, niclosamide.