Atovaquone: An Antiprotozoal Drug Suppresses Primary and Resistant Breast Tumor Growth by Inhibiting HER2/β-Catenin Signaling

Abstract

Breast cancer is the second leading cause of cancer-related mortality in women. In the current study, we evaluated the anticancer effects of an antiprotozoal drug, atovaquone, against several breast cancer cell lines. Our results showed that atovaquone treatment induced apoptosis and inhibited the growth of all the breast cancer cell lines tested, including several patient-derived cells. In addition, atovaquone treatment significantly reduced the expression of HER2, β-catenin, and its downstream molecules such as pGSK-3β, TCF-4, cyclin D1, and c-Myc in vitro Efficacy of atovaquone was further evaluated in an in vivo tumor model by orthotropic implantation of two highly aggressive 4T1 and CI66 breast cancer cells in the mammary fat pad of female mice. Our results demonstrated that oral administration of atovaquone suppressed the growth of CI66 and 4T1 tumors by 70% and 60%, respectively. Paclitaxel is the first-line chemotherapeutic agent for metastatic breast cancer. We demonstrate that atovaquone administration suppressed the growth of 4T1 paclitaxel-resistant tumors by 40%. Tumors from atovaquone-treated mice exhibited reduced HER2, β-catenin, and c-Myc levels alongside an increase in apoptosis in all the three tumor models when analyzed by Western blotting, IHC, and TUNEL assay. Taken together, our results indicate that atovaquone effectively reduces the growth of primary and paclitaxel-resistant breast tumors. Atovaquone is already in the clinics with high safety and tolerability profile. Therefore, the findings from our studies will potentially prompt further clinical investigation into repurposing atovaquone for the treatment of patients with advanced breast cancer.

Figure 1. Atovaquone exhibits potent anti-tumor activity in breast cancer cell lines.

A) HCC1806, B) SKBR3, C) MCF-7, D) 4T1, E) T47D, F) CI66 cells were treated with increasing concentrations of atovaquone for 24, 48 and 72h. Cell survival was measured with sulforhodamine B (SRB) assay to estimate the IC₅₀ values. Patient derived cell lines G) TX-BR-237 and H) TX-BR-290 were also evaluated for cytotoxic effect of atovaquone. The experiments were repeated at least three times with 8 replicates in each experiment. Colony formation assay was performed by seeding 400–600 cells/well. 4T1 cells were fixed and stained using crystal violet (0.5%) after 9 days and MCF-7 cells after 14 days. Number of colonies formed in control and atovaquone treated wells were quantitated using Image J software. Representative Images of colonies and their quantification in (I-J) 4T1 cells and (K-L) MCF-7 cells. Data shown as mean ± SD; (n=3). Student’s t test for unpaired samples was used to perform statistical comparisons.

Figure 2. Atovaquone induces apoptosis in breast cancer cells.

A) SKBR3, B) HCC1806, C) 4T1, D) MCF-7 cells was treated with or without atovaquone (10, 20 and 30 µM) for 72h. Cells were then collected and labeled with Annexin V FITC. Cells that were positive for Annexin or PI or both were measured using flowcytometry. Each experiment was repeated more than three times independently. E) Western blots analyses representing the basal level of HER2 and β-catenin in breast cancer cells. *Statistically different when compared with control (p<0.05).

Figure 3. Atovaquone down regulates HER2/β-catenin signaling in breast cancer cells.

A) SKBR3, B) 4T1, C) MCF-7 and D) HCC1806, cells were treated with varying concentrations of atovaquone for 72h. Representative blots showing the concentration dependent effect of atovaquone on p-HER2, HER2, β-catenin, p-GSK3β, GSK3β c-Myc, TCF-4, TCF-1 cyclin D1, MMP-7, cleaved caspase 3, cleaved PARP. Actin was used as loading control. Each experiment was repeated three times independently.

Figure 4. Change in HER2 expression modulates the effect of atovaquone.

A) Percentage cell survival of MCF-7HH cells when treated with atovaquone at indicated concentrations and time points. B) MCF-7 and MCF-7HH cells were treated with 10, 20 and 30 μM atovaquone for 72h and whole lysate was analyzed by western blotting for HER2, β-catenin, p-GSK3β, c-Myc, TCF-4, cyclin D1, MMP-7, cleavage of caspase 3 and PARP. C) SKBR3 cells transfected with either scrambled or HER2 siRNA and treated with or without 20µM atovaquone for 72h. Whole cell lysate was evaluated for HER2, β-catenin, c-Myc, cyclin D1, cleavage of caspase 3 & PARP. MCF-7HH were treated with atovaquone (20 μM) for 72 hours after transfecting the cells with D) HER2 siRNA or scrambled siRNA and E) HER2 shRNA or scrambled shRNA. Levels of HER2, β-catenin, c-Myc, cyclin D1, cleaved caspase 3, cleaved PARP were evaluated by Western blotting. Blots were quantitated and normalized with actin. Each experiment was repeated three times independently. (F) MCF-7HH cells treated with 10μM MG-132 and 20μM atovaquone alone and in combination for 72 hours. β-catenin was immunoprecipitated from control and 20 μM atovaquone-treated cells and probed for HER2 in (G) MCF-7 and (H) MCF-7HH cells. IgG was used as loading control.

Figure 5. Atovaquone suppresses the growth of 4T1 and CI66 breast tumors and inhibits HER2/β-catenin signaling in vivo.

A) About 0.07 × 10⁶ 4T1 and 0.1X10⁶ CI66 breast cancer cells were injected orthotropically in the right and left mammary fat pads of 4 to 6 weeks old Balb/c female mice. Mice were given 30 mg/kg of atovaquone by oral gavage every day till day 25. A) Tumor growth curve in 4T1 cells. Values were plotted as mean ± SEM. B) Orthotropically implanted 4T1 tumors were removed aseptically after terminating the experiments. Tumors were homogenized, lysed, and analyzed for HER2, β-catenin, c-Myc, c-caspase-3 and c-PARP. Blots were stripped and reprobed with actin antibody to verify equal protein loading; each lane of blot represents tumor from individual mouse. C) Tumors were sectioned and immunostained for HER2, β-catenin and c-caspase-3. D) Representative images from control and atovaquone treated 4T1 tumors by TUNEL assay. E) Tumor growth curve in CI66 cells. Values were plotted as mean ± SEM. F) Effect of atovaquone on CI66 tumor weight. G) CI66 tumors were minced, lysed and analyzed for HER2, β-catenin, c-Myc, and c-caspase-3 and c-PARP. Each band represents tumor from individual mouse. H) Weight of the mice during the study.

Figure 6. Atovaquone inhibits the growth of paclitaxel-resistant cells in vitro and in vivo by inhibiting HER2/β-catenin signaling.

A) Percentage cell survival of 4T1PR cells when treated with atovaquone at indicated concentrations and time points. B) Dose and time-dependent increase in apoptotic cell population after treatment with atovaquone in 4T1PR cells as analyzed by AnnexinV/PI co-staining. Reduction in (C) 4T1PR breast tumor volume and (D) tumor weight after treatment with 25 mg/Kg atovaquone. (E) Expression of HER2, β-catenin, c-PARP and c-caspase-3 in tumor lysates, each blot indicates tumor lysate from individual mouse. (F) Bar graph representation of quantified blots. G) Tumors from control and atovaquone treated mice were dissected out and kept in 4% formalin solution. Tumors were then sliced about 5–10 μm thick and placed on glass slides. Treated and untreated tumors were stained with HER2, β-catenin and c-caspase-3 antibodies. H) Representative images from control and atovaquone treated 4T1PR tumors by TUNEL assay.