WNT/beta-catenin mediates radiation resistance of mouse mammary progenitor cells

Abstract

Recent studies have identified a subpopulation of highly tumorigenic cells with stem/progenitor cell properties from human breast cancers, and it has been suggested that stem/progenitor cells, which remain after breast cancer therapy, may give rise to recurrent disease. We hypothesized that progenitor cells are resistant to radiation, a component of conventional breast cancer therapy, and that that resistance is mediated at least in part by Wnt signaling, which has been implicated in stem cell survival. To test this hypothesis, we investigated radioresistance by treating primary BALB/c mouse mammary epithelial cells with clinically relevant doses of radiation and found enrichment in normal progenitor cells (stem cell antigen 1-positive and side population progenitors). Radiation selectively enriched for progenitors in mammary epithelial cells isolated from transgenic mice with activated Wnt/beta-catenin signaling but not for background-matched controls, and irradiated stem cell antigen 1-positive cells had a selective increase in active beta-catenin and survivin expression compared with stem cell antigen 1-negative cells. In clonogenic assays, colony formation in the stem cell antigen 1-positive progenitors was unaffected by clinically relevant doses of radiation. Radiation also induced enrichment of side population progenitors in the human breast cancer cell line MCF-7. These data demonstrate that, compared with differentiated cells, progenitor cells have different cell survival properties that may facilitate the development of targeted antiprogenitor cell therapies.

Fig. 1.

Clinically relevant doses of radiation increased the percentage of progenitor cells (%SP and Sca1⁺) in primary MEC culture and human MCF-7 cells. (A) MECs were isolated from BALB/c mice, cultured for 3 days, irradiated, and analyzed for %SP by Hoechst 33342 staining and flow cytometry. Radiation selectively increased the progenitor fraction (%SP) (P = 0.015 for 2 Gy, 0.008 for 4 Gy, and 0.05 for 6 Gy by the two-tailed t test). (B) MCF-7 cells were analyzed for %SP by Hoechst 33342 staining and flow cytometry. Radiation selectively increased the progenitor fraction (%SP) (P = 0.05 for 0 Gy vs. 4 Gy by the two-tailed t test). (C) Cells were analyzed for Sca1 in the SP 24 h after irradiation. Radiation selectively increased the Sca1⁺ (progenitor) fraction within the SP by killing the more sensitive Sca1⁻ (nonprogenitor) cells (P < 0.05 for Sca1⁺ to Sca1⁻ at 0 Gy vs. 2–8 Gy). The differences in effects of doses of 2 Gy vs. higher doses were not significant. (D) Anesthetized BALB/c mice were immobilized supine, and mammary glands (entire ventral surface) were irradiated. MECs were isolated 48 h after irradiation and analyzed immediately for Sca1 by flow cytometry. Radiation selectively increased the Sca1⁺ (progenitor) fraction and decreased the Sca1⁻ (nonprogenitor) cells. ∗, P < 0.0001.

Fig. 2.

In vivo radiation increased the percentage of CD24⁺CD29⁺ positive cells from MCF-7 cells but not uncultured MECs. (A) Freshly digested MECs were analyzed for lin⁻CD24⁺CD29⁺ 48 h after in vivo irradiation. The CD24⁺CD29⁺ population is sensitive to radiation. (B) MCF-7 cells were irradiated and analyzed for lin⁻CD24⁺CD29⁺ by flow cytometry. Radiation selectively decreased the lin⁻CD24⁺CD29^lo fraction cells (P = 0.003 for 0 Gy vs. 2 Gy, and P = 0.0002 for 0 Gy vs. 4 Gy).

Fig. 3.

Radiation induced more DNA damage foci in Sca1⁻ cells 2 h after irradiation. Sca1⁺ and Sca1⁻ cells from BALB/c MECs were sorted onto glass slides after irradiation with 2 Gy and immunostained with anti-phospho-H2AX. (Scale bar: 10 μm.) There were significantly more DNA-damaged foci in the Sca1⁻ population than in the Sca1⁺ population (3.7-fold difference, ∗∗, P < 0.05).

Fig. 4.

Clinically relevant doses of radiation led to an increased percentage of SP cells in primary mouse MECs isolated from mice with a gain-of-function, conditionally stabilized β-catenin allele and from Wnt-1 transgenic mice compared with in control cells. (A) MECs from Wnt-1 transgenic mice at 16 weeks of age and wild-type mice of the same background were stained with Hoechst 33342, and the %SP was analyzed by using flow cytometry as described. ∗, P = 0.08 for 0 Gy Wnt vs. 2 Gy Wnt, P = 0.001 for 0 Gy wild type vs. Wnt, and P = 0.04 for 2 Gy wild type vs. Wnt by two-tailed t test. MECs from mice treated with AdCre recombinase to generate stabilized β-catenin or an AdLacZ control vector were stained with Hoechst 33342, and the %SP was analyzed by using flow cytometry as described (P < 0.05 for 0 Gy vs. 2 Gy, and P < 0.05 for 0 Gy vs. 4 Gy). Radiation selectively activated β-catenin and survivin in Sca1⁺ cells. (B) Quantitative assessment of activated β-catenin signaling was assessed by flow cytometry after staining for Sca1 and unphosphorylated β-catenin. Real-time PCR for survivin expression was performed 24 h after irradiation in Sca1⁺ and Sca1⁻ cells.