Atovaquone Suppresses Triple-Negative Breast Tumor Growth by Reducing Immune-Suppressive Cells

Abstract

A major contributing factor in triple-negative breast cancer progression is its ability to evade immune surveillance. One mechanism for this immunosuppression is through ribosomal protein S19 (RPS19), which facilitates myeloid-derived suppressor cells (MDSCs) recruitment in tumors, which generate cytokines TGF-β and IL-10 and induce regulatory T cells (Tregs), all of which are immunosuppressive and enhance tumor progression. Hence, enhancing the immune system in breast tumors could be a strategy for anticancer therapeutics. The present study evaluated the immune response of atovaquone, an antiprotozoal drug, in three independent breast-tumor models. Our results demonstrated that oral administration of atovaquone reduced HCC1806, CI66 and 4T1 paclitaxel-resistant (4T1-PR) breast-tumor growth by 45%, 70% and 42%, respectively. MDSCs, TGF-β, IL-10 and Tregs of blood and tumors were analyzed from all of these in vivo models. Our results demonstrated that atovaquone treatment in mice bearing HCC1806 tumors reduced MDSCs from tumor and blood by 70% and 30%, respectively. We also observed a 25% reduction in tumor MDSCs in atovaquone-treated mice bearing CI66 and 4T1-PR tumors. In addition, a decrease in TGF-β and IL-10 in tumor lysates was observed in atovaquone-treated mice with a reduction in tumor Tregs. Moreover, a significant reduction in the expression of RPS19 was found in tumors treated with atovaquone.

Keywords: C5aR1; HIF1α; RPS19; atovaquone; cytokines; myeloid-derived tumor-suppressor cells; regulatory T cells; repurposing; triple-negative breast cancer.

Figure 1

Atovaquone suppresses breast tumor growth. Human peripheral blood mononuclear cells (8 × 10⁶) cells were injected intraperitoneally 7 days after HCC1806 tumor cells’ implantation in NCG/SCID mice; n = 5/group. (A) HCC1806 tumor growth curves. (B) Endpoint tumor weights. (C) Tumor growth curve of CI66 cells; n = 6/group. (D) Bar graph representing average tumor volume of control and atovaquone-treated mice at day 25; 4T1 paclitaxel-resistant cells were implanted in right and left flanks of female Balb/c mice, and treatment was 25 mg/kg atovaquone daily; n = 10/group. (E) Tumor growth curve. (F) Tumor weight from control and atovaquone-treated group at day 20. Values were plotted as mean ± SEM. Statistically significant at p < 0.05 when compared with control.

Figure 2

Effect of Atovaquone on MDSCs. Peripheral blood mononuclear cells (PBMCs) were collected from the blood obtained from HCC1806 tumor-bearing mice. (A) Bar chart representing percent MDSCs which were stained with CD11b and CD33 human antibodies; n = 4. Tumors from control and atovaquone-treated group in all three in vivo experiments were isolated, processed and suspended into single-cell suspension with the help of gentle MACS dissociator using tumor dissociator kit. Percentage of MDSCs from (B) HCC1806 tumor cells (stained with CD11b/CD33 human antibodies); n = 3 (C) CI66 tumors (stained with CD11b/Gr-1 mouse antibodies); n = 3 and (D) 4T1-PR tumors (stained with CD11b/Gr-1 mouse antibodies); n = 3. Data shown as mean ± SEM. A p-value less than 0.05 was considered to be significant.

Figure 3

Atovaquone suppresses regulatory T cells (Treg) in HCC1806 and 4T1-PR tumors. Tumors were removed from mice bearing HCC1806 and 4T1-PR tumors at the end of the experiment. Tumors from control and atovaquone-treated groups were processed and suspended into single-cell suspension, using a tumor dissociator kit. Modulation of Treg cells was monitored by immunostaining with CD120b/CD4 and analyzed by flow cytometry. (A) Percent Tregs from control and atovaquone-treated groups stained with CD120b in HCC1806 tumor cells. (B) %Tregs double stained with CD120b and CD4 mouse specific antibodies in 4T1PR tumor cells. Values were plotted as mean ± SEM from n = 4 samples.

Figure 4

Reduction of immune-suppressive cytokines with atovaquone treatment. Equal amount of protein from HCC1806, as well as CI66 tumors from control and atovaquone-treated mice, was used to perform ELISA assay for TGF-β and IL-10. Bar graph representing (A) TGF-β (HCC1806 tumor lysate), (B) IL-10 (HCC1806 tumor lysate) and (C) TGF-β (CI66 tumor lysate) levels from control and treatment groups. Data shown as mean ± SEM from at least three individual tumor samples. Statistically significant at p < 0.05 when compared with control.

Figure 5

Reduction in the expression of RPS19 with increased apoptosis after atovaquone treatment. Tumors were isolated from mice bearing HCC1806 tumors at the day of termination. A part of tumor was fixed in formalin for immunohistochemical analysis, as well as for Western blot analysis. (A) Treated and untreated HCC1806 tumor sections were analyzed for RPS19 by IHC. (B) Western blot analyses showing the expression of RPS19 in control and treated HCC1806 tumors. (C) Expression of C5aR1 in atovaquone-treated and untreated HCC1806 tumors. (D) HIF-1α expression in HCC1806 cells treated with or without atovaquone (ATQ) under both normoxic and hypoxic conditions by Western blotting. (E) Representative images of excised tumor sections from control and atovaquone-treated group analyzed by TUNEL assay. Each section indicates a tumor from an individual mouse.

Figure 6

Schematic diagram depicting the role of atovaquone in immune system.