In Vitro and In Vivo Efficacy of the Monocarboxylate Transporter 1 Inhibitor AR-C155858 in the Murine 4T1 Breast Cancer Tumor Model

Abstract

Monocarboxylate transporter 1 (MCT1), also known as a L-lactate transporter, is a potential therapeutic target in cancer. The objectives of this study were to evaluate efficacy and assess concentration-effect relationships of AR-C155858 (a selective and potent MCT1 inhibitor) in murine 4T1 breast cancer cells and in the 4T1 tumor xenograft model. Western blotting of 4T1 cells demonstrated triple negative breast cancer (TNBC) characteristics and overexpression of MCT1 and CD147 (a MCT1 accessory protein), but absence of MCT4 expression. AR-C155858 inhibited the cellular L-lactate uptake and cellular proliferation at low nanomolar potencies (IC₅₀ values of 25.0 ± 4.2 and 20.2 ± 0.2 nM, respectively). In the xenograft 4T1 mouse model of immunocompetent animals, AR-C155858 (10 mg/kg i.p. once daily) had no effect on tumor volume and weight. Treatment with AR-C155858 resulted in slightly increased tumor lactate concentrations; however, the changes were not statistically significant. AR-C155858 was well tolerated, as demonstrated by the unchanged body weight and blood lactate concentrations. Average blood and tumor AR-C155858 concentrations (110 ± 22 and 574 ± 245 nM, respectively), 24 h after the last dose, were well above the IC₅₀ values. These data indicate that AR-C155858 penetrated 4T1 xenograft tumors and was present at high concentrations but was ineffective in decreasing tumor growth. Evaluations of AR-C155858 in other preclinical models of breast cancer are needed to further assess its efficacy.

Keywords: 4T1; AR-C155858; breast cancer; monocarboxylate transporter 1.

Fig. 1

Chemical structure of AR-C155858 (6-[(3,5-dimethyl-1H-pyrazol-4-yl)methyl]-5-[(4S)-4-hydroxy-1,2-oxazolidine-2-carbonyl]-3-methyl-1-(2-methylpropyl)thienol[2,3-d]pyrimidine-2,4-dione)

Fig. 2

Characterization of the murine 4T1 breast tumor model. The murine 4T1 breast tumor model exhibited similar TNBC characteristics as in human TNBC. Presented are cytosolic ER (a) and PR (b) expressions, plasma membrane expression of HER2 (c), plasma membrane protein expression of MCT1 (d), and CD147 (e) and MCT4 (f) proteins in 4T1 cells relative to murine mammary epithelial cells (NMuMG). Histograms show corresponding densitometry analysis of the membranes from western blots of MCT1, MCT4, and CD147 (20 μg per lane). GAPDH and Na⁺K⁺ ATPase were used as loading controls for total and membrane protein, respectively

Fig. 3

l-Lactate uptake kinetics in 4T1 cells. Presented is the time-dependent l-lactate uptake at pH 6.0 (a), and concentration-dependent uptake of l-lactate at pH 6.0 (b). The concentration-dependent data were best fitted to a Michaelis-Menten equation with diffusional uptake clearance component (P). Observed mean data from three studies are shown (symbols), with the line representing the model-fitted results

Fig. 4

Effect of AR-C155858 on l-lactate uptake and cell growth in 4T1 cells. Inhibition of radiolabeled l-lactate (0.5 mM) uptake at pH 6.0 by AR-C155858 (a). Percentage of cell growth after 48 h of AR-C155858 treatment with results normalized to vehicle control-treated cells (b). Observed mean data from three studies are shown (symbols), with the line representing model fitting results

Fig. 5

In vivo efficacy of AR-C155858 in the 4T1 breast tumor xenograft. A schematic representation of the experimental timeline and dosing schedule following orthotopic inoculation of 4T1 cells subcutaneously into the mammary fat pad of female BALB/c mice. Mice were given i.p. injection of AR-C155858 (10 mg/kg) or vehicle control once a day (a). Body weight (b) and tumor volume (c) were monitored throughout the study. Tumor weight (d), intratumor (e), and blood (f) lactate concentration were measured at the end of the treatment. Primary tumor excised at the end of the study showed tumor necrosis in both vehicle- and AR-C155858-treated tumors (g). All the data are presented as mean ± SD, n = 5 in each group. NS nonsignificant, P > 0.05 compared to the vehicle control-treated group (two-way ANOVA followed by Bonferroni’s post-hoc test or nonparametric Student’s t test)

Fig. 6

Blood and total tumor AR-C155858 concentrations measured at the end of the treatment (24 h after the last AR-C155858 dose). Assuming tumor density of 1 g/cm3, tumor AR-C155858 concentrations were normalized to gram of tumor tissue and expressed in nanomolar. All the data are presented as mean ± SD, n = 5. **P < 0.01, compared to the blood AR-C155858 concentration (nonparametric Student’s t test)