Repurposing mebendazole against triple-negative breast cancer leptomeningeal disease

Abstract

Purpose: Triple-negative breast cancer (TNBC) is an aggressive subtype that often metastasizes to the brain. Leptomeningeal disease (LMD), a devastating brain metastasis common in TNBC, has limited treatment options. We sought to test whether the common anti-helminthic drug mebendazole (MBZ) may be effective against murine TNBC LMD.

Methods: A small-molecule screen involving TNBC cell lines identified benzimidazoles as potential therapeutic agents for further study. In vitro migration assays were used to evaluate cell migration capacity and the effect of MBZ. For in vivo testing, LMD was introduced into BALB/c athymic nude mice through internal carotid artery injections of brain-tropic MDA-MB-231-BR or MCF7-BR cells. Tumor growth and spread was monitored by bioluminescence imaging. MBZ was given orally at 50 and 100 mg/kg doses. MBZ bioavailability was assayed by mass spectrometry.

Results: Bioinformatic analysis and migration assays revealed higher migratory capacity of TNBC compared to other breast cancer subtypes. MBZ effectively slowed down migration of TNBC cell line MDA-MB-231 and its brain tropic derivative MDA-MB-231-BR. In animal studies, MBZ reduced tumor growth and extended survival in the LMD model produced by MDA-MB-231-BR cells. MBZ did not have an effect in the non-migratory MCF7-BR model.

Conclusions: We demonstrated that MBZ is a safe and effective oral agent in an animal model of TNBC LMD. Our findings are concordant with previous efforts involving MBZ and central nervous system pathology and further support the drug's potential utility as an alternative therapeutic for TNBC LMD.

Keywords: Breast cancer; drug repurposing; leptomeningeal disease; mebendazole.

Figure 1. Benzimidazoles as a potential treatment for migratory cancers, such as TNBC.

(A) Small molecule screen used to identify pharmacologic compounds active against triple-negative breast cancer (TNBC) cell line SUM149PT, but not against a non-tumorigenic breast cell line MCF10a [22]. (B) Benzimidazoles may be more selective against TNBC. Shown are compounds more effective against metastatic TNBC cell line MDA-MD-231 compared to a metastatic luminal A breast cancer cell line MCF7. (A, B) Diagonal line is placed for agents equally effective against indicated cell lines. Benzimidazoles are labeled in red. (C) GO enrichment terms associated with migration are overrepresented in nematodes and underrepresented in flatworms [32]. (D) Disruption of ligand-receptor interactions important for cell migration and LMD represent a common mode of benzimidazole action in mammalian cells. Results of the Drug-Path database query [35], which shows the pathways significantly affected in mammalian cells by benzimidazoles. The pathways strongly associated with cell migration (in red color) were affected by the majority of tested benzimidazoles [albendazole (A), fenbendazole (F), mebendazole (M), nocodazole (N), parmendazole (N), and thiabendazole (T)] and have a low false discovery rate (FDR).

Figure 2. Mebendazole (MBZ) as a potential treatment against leptomeningeal disease.

(A, B) Sensitivity to MBZ of TNBC cell line MDA-MB-231, hormone receptor positive cell line MCF7 (A), and their brain-tropic derivatives MDA-MB-231-BR and MCF7-BR (B). Brain-tropic MDA-MB-231-BR cell line is slightly more resistant to MBZ than the parental cell line MDA-MB-231: MDA-MB-231-BR (IC₅₀=0.16 μM), MDA-MB-231 (IC₅₀=0.14 μM), MCF7-BR (IC₅₀=0.19 μM), and MCF7 (IC₅₀ = 0.19 μM). (C) Infra-red spectra (FT-IR) of MBZ polymorphs revealing the presence of MBZ polymorph C (MBZ-C) and polymorph B (MBZ-B) in the MBZ from Sigma, CAS # 31431–39-7. (D) MBZ given at an oral dose of 100 mg/kg reaches therapeutic concentrations in the cerebrospinal fluid (CSF) of NuNu mice (median [MBZ] = 105.9 ng/ml ~ 0.36 μM). Plasma MBZ concentrations represent total quantity of MBZ, and CSF concentrations represent free, unbound MBZ. A horizontal line at 59 ng/mL corresponds to the IC₅₀ = 0.20 μM of MBZ. MDA231 = MDA-MB-231. MDA231-BR = MDA-MB-231-BR. Significance: ***, p < 0.001, NS = not significant.

Figure 3. MBZ reduces the migration of TNBC MDA-MB-231 and MDA-MB-231-BR cells.

(A, B) TNBCs have higher migratory capacity compared to other breast cancer subtypes. Migration scores from Nair et al. [7] were compared among breast cancer subtypes in TCGA patient data (A) and cell lines (B). (C, D) MDA-MB-231 and MDA-MB-231-BR have higher migration capabilities than MCF7 and MCF7-BR cells. Migratory capability of MDA-MB-231 cells increases upon acquiring brain-tropic status and is effectively inhibited by MBZ. Neither MCF-7 nor MCF-7-BR migrated significantly during 20 h. (C) Representative inverse fluorescence images of DAPI-stained membranes from Boyden chamber during 20 h. DAPI-stained cell nuclei are shown as dark gray spots in the background of white membrane pores. Scale bar: 200 μm. (D) Quantitation of migration in MCF7, MDA-MB-231, and brain-tropic MCF7-BR and MDA-MB-231-BR cells from (C). (E) Survival during 20 h treatment with MBZ. Plating efficiencies of untreated cell lines were not significantly different. MDA231 = MDA-MB-231, MDA231-BR = MDA-MB-231-BR. FOV = field of view. Significance analysis: ANOVA, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001.

Figure 4. The model of leptomeningeal metastasis.

(A) Schematics of an internal carotid artery injection of tumor cells to establish a murine model of LMD. (B) H&E-stained section from mouse brain affected by LMD. Arrows point to cancer cells in leptomeningeal space. (C) Patient brain T1W+C MRI sequence shows the anatomical location of leptomeningeal disease (LMD) (red arrow). (D-H) Immunofluorescence images depict dissemination of neoplastic cells into leptomeningeal space. BP = brain parenchyma. Arrows point to cancer cells in leptomeningeal space. (D, E) Vimentin, a marker of epithelial-to-mesenchymal transition, is highly expressed in MDA-MB-231 cells. Antibody against human vimentin (hVim) identifies MDA-MB-231-BR breast cancer cells. (F) Antibody against pan-cytokeratin (PanCK) identifies MCF7-BR breast cancer cells. (E, F) Antibody against laminin (Lam) shows the location of pia. (G) Bioluminescence images reveal intracranial disease and spinal dissemination (red arrows). (H) Spinal metastases identified by bioluminescence were verified by subsequent H&E staining. Right panel is a magnified version of a region indicated in the left panel. Black arrows point to the same spinal metastasis in the 4x image and a magnified (x20) image.

Figure 5. MBZ is effective against TNBC LMD in a mouse model.

(A) Experimental timeline. (B) Representative bioluminescence images of mice with LMD in control and treatment groups (at 50 and 100 mg/kg). (C, D) MBZ slows metastatic growth as detected by bioluminescence imaging (C) and improves survival (D) in a mouse model of TNBC LMD formed by the injection of MDA-MB-231-BR cells into internal carotid artery of NuNu mice. (E, F) MBZ (50 mg/kg) shows no effect on metastatic growth (E) and survival (F) in the MCF7-BR model of LMD. Significance: *, p < 0.05, ***, p < 0.001, NS = not significant. Experiments in (C, E) were analyzed using repeated-measures method. Post hoc pairwise comparisons were performed using a Tukey adjustment for multiple comparisons.