Abstract 3999: Intracellular redox status determines self-renewal activity of breast cancer stem-like cells through activation of Nrf2-mediated FoxO3a-Bmi-1 signaling

Abstract

Abstract A subpopulation of cancer cells, termed cancer stem cells (CSCs), has a self-renewal property, which drives cancer recurrence and tumor resistance. It has been reported that CSCs possess enhanced capabilities to protect against oxidative stress caused by reactive oxygen species (ROS) compared with non-stem-like cancer cells. In the present study, we found that the redox-sensitive transcription factor NF-E2-related factor 2 (Nrf2) plays a role in self-renewal activity of human breast cancer cells, and that this was mediated by Bmi-1. The manifestation of stemness in breast CSC-like cells was associated with an elevated ratio of reduced to oxidized glutathione (GSH) and consequently, lowered ROS levels compared with parent breast cancer (MCF-7) cells. We observed that there was a concurrent increase in the expression of CD44, glutamate cysteine ligase (GCLC) and Bmi-1 in CD24low/CD44high breast cancer stem-like cells sorted from MCF-7 mammospheres. Treatment with buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis, led to a decrease in the number and the size of mammospheres, which was accompanied by the reduction of Bmi-1 expression. Conversely, treatment with a membrane permeable GSH ethyl ester (GSH-MEE) enhanced the size and the number of mammospheres. In addition, phosphorylation of AMPK (Thr 172) and FoxO3a (Ser 7) as well as expression of Bmi-1 was increased in mammosphere cells treated with GSH-MEE. When GSH-MEE-treated mammospheres were challenged with hydrogen peroxide, the phosphorylation of AMPK was abolished. Notably, FoxO3a directly bound to the Bmi-1 promoter as assessed by the chromatin immunoprecipitation assay. Moreover, elevated nuclear accumulation of Nrf2 protein was evident in cells growing under sphere-forming conditions. Under physiologic conditions, Nrf2 is sequestered in the cytoplasm as an inactive complex with Keap1. Sphere-forming breast cancer cells exhibited an enhanced interaction between Keap1 and CD44, which may account for an increased translocation of Nrf2 in nucleus. We found that expression of GCLC, FoxO3a and Bmi-1 was inhibited following introduction of Nrf2 siRNA into MCF-7 mammsophere cells. Knockdown of Nrf2 suppressed the xenograft growth of orthotopically injected breast cancer stem-like cells in athymic nude mice, and this was accompanied by decreased expression of GCLC, FoxO3a and Bmi-1. In conclusion, CD44 promotes Nrf2 accumulation in breast cancer stem-like cells, which, in turn, upregulates GCLC expression and consequently enhances GSH biosynthesis with concomitant reduction in intracellular ROS levels. The consequent upregulation of nuclear FoxO3a and its binding to the Bmi-1 promoter may stimulate self-renewal and growth of breast cancer stem-like cells. Citation Format: Do-Hee Kim, Jeong-Hoon Jang, Young-Joon Surh. Intracellular redox status determines self-renewal activity of breast cancer stem-like cells through activation of Nrf2-mediated FoxO3a-Bmi-1 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3999.