The antihelmintic drug pyrvinium pamoate targets aggressive breast cancer

Abstract

WNT signaling plays a key role in the self-renewal of tumor initiation cells (TICs). In this study, we used pyrvinium pamoate (PP), an FDA-approved antihelmintic drug that inhibits WNT signaling, to test whether pharmacologic inhibition of WNT signaling can specifically target TICs of aggressive breast cancer cells. SUM-149, an inflammatory breast cancer cell line, and SUM-159, a metaplastic basal-type breast cancer cell line, were used in these studies. We found that PP inhibited primary and secondary mammosphere formation of cancer cells at nanomolar concentrations, at least 10 times less than the dose needed to have a toxic effect on cancer cells. A comparable mammosphere formation IC50 dose to that observed in cancer cell lines was obtained using malignant pleural effusion samples from patients with IBC. A decrease in activity of the TIC surrogate aldehyde dehydrogenase was observed in PP-treated cells, and inhibition of WNT signaling by PP was associated with down-regulation of a panel of markers associated with epithelial-mesenchymal transition. In vivo, intratumoral injection was associated with tumor necrosis, and intraperitoneal injection into mice with tumor xenografts caused significant tumor growth delay and a trend toward decreased lung metastasis. In in vitro mammosphere-based and monolayer-based clonogenic assays, we found that PP radiosensitized cells in monolayer culture but not mammosphere culture. These findings suggest WNT signaling inhibition may be a feasible strategy for targeting aggressive breast cancer. Investigation and modification of the bioavailability and toxicity profile of systemic PP are warranted.

Figure 1. PP targeting of the WNT/β-catenin pathway.

(A) PP decreased the protein levels of nonphospho-β-catenin, an active form of β-catenin, and β-catenin in SUM-149 and SUM-159 cell lines. Data shown are representative of three independent experiments. (B) PP also decreased the GFP-positive population in SUM-159 cells transduced with a TOP-GFP construct. (C) Summary of three independent GFP-detection experiments.

Figure 2. The toxic effects of PP on breast cancer cells.

SUM-149 and SUM-159 were treated with increasing concentrations of PP for 96 hours. The toxic effect of PP was measured by MTS assay (A, B) or cell counting by flow cytometry (C). The data shown are representative or summary of three independent experiments.

Figure 3. Inhibitory effect of PP on mammosphere formation.

(A, B) Primary mammosphere formation. SUM-149 (A) and SUM-159 (B) cells were incubated with indicated doses of PP or DMSO in mammosphere formation media for 7 days. PP treatment reduced the number of primary mammospheres in a dose-dependent manner. (C, D) Secondary mammosphere formation. SUM-149 (C) and SUM-159 (D) cells were grown in mammosphere formation media and treated with indicated doses of PP for 4 days. Then primary spheres were trypsinized and seeded as secondary mammospheres. [Add sentence summarizing effect of PP.] The results shown are representative of three independent experiments.

Figure 4. PP decreased the ALDH-positive cell population.

SUM-159 cells were treated with PP (1 nM) or DMSO for 96 hours and subjected to an Aldefluor assay and flow cytometry analysis. (A) A set of representative flow cytometry dot plots. DEAB served as a negative control. (B) PP decreased the percentage of ALDH-positive cells. The data shown are the means of three independent experiments.

Figure 5. PP inhibited tumor growth in vivo when it was administered intraperitoneally.

SUM-159 cells were injected into the mammary fat pads of SCID/Beige mice. Once tumors grew to approximately 100 mm³, either vehicle (DMSO/saline 50%/50%) or PP was injected IP three times per week. The dose of PP was scaled up according to the following regimen: 1^st dose: 0.1 mg/kg; 2^nd dose: 0.2 mg/kg; 3^rd dose: 0.4 mg/kg; 4^th dose: 0.6 mg/kg; 5^th dose: 0.8 mg/kg; and 6^th dose: 1.0 mg/kg. After the 6^th injection, the dose was kept at 1 mg/kg. PP delayed tumor growth beginning at 4 days of the initial treatment. There were 11 mice in the treatment group and 10 mice in the control group. The Student t-test was used to calculate P value. Error bars indicate standard deviation.

Figure 6. PP inhibited mammosphere formation by primary breast cancer cells.

Cancer cells were purified from pleural effusion samples as described. The cells were seeded at 40,000/ml in mammosphere formation media. (A) PP treatment reduced the number of primary mammospheres in a dose-dependent manner. (B) Flow cytometry revealed a decrease in ALDH-positive activity after PP treatment.

Figure 7. EMT markers were down-regulated in SUM-149 and SUM-159 cells treated with PP.

SUM-149 and SUM-159 cells were treated with different doses of PP for 96 hours followed by Western blotting analysis using antibodies targeting different EMT markers. Slug, N-cadherin, and vimentin were down-regulated by 10 nM PP. The results shown are representative of three independent experiments.

Figure 8. PP sensitized SUM-149 cells to radiation therapy.

SUM-149 cells cultured in normal media were treated without or with 2 nM PP for 96 hours, followed by treatment with the indicated dose of radiation. Then cells were trypsinized and seeded in mammosphere media (A) or monolayer media (B). The results are representative of at least three independent experiments.