Abstract 3306: The RNA-binding protein HuR radiosensitizes human TNBC cells by modulating the cellular response to DNA damage and oxidative stress

Abstract

Abstract The RNA-binding protein human antigen R (HuR) associates with U-/AU-rich mRNAs encoding proteins that control cell proliferation, metabolism and the stress response. HuR is overexpressed in several human cancers and its overexpression is associated with poor prognosis and resistance to therapy. While the role of HuR in drug resistance has been studied, its contribution to radiation resistance has not been examined. Therefore, we investigated the role of HuR in radiation resistance of triple negative breast cancer (TNBC) cells: MDA-MB-231, MDA-MB-468 and Hs578t. Reduction of HuR expression using small interfering (si) RNA decreased cell proliferation and sensitized TNBC cells to ionizing radiation. Clonogenic assays indicated that silencing HuR suppressed the clonogenic survival of all three TNBC cell lines with survival at 2 Gy (SF2) reduced from 59%, 49%, 65% in control cells to 40%, 33%, and 46% in siHuR-treated MDA-MB-231, MDA-MB-468 and Hs578t cells, respectively. To delineate the underlying mechanism of radiosensitization and to identify candidate mRNAs showing altered levels after silencing HuR, we undertook a ribonomic approach. First, since ionizing radiation enhances the production of reactive oxygen species (ROS), causing DNA damage, we investigated the possible involvement of ROS in siHuR-mediated radiosensitization. ROS production in control or HuR-silenced cells treated with or without radiation was measured using the fluorescent dye 2′-7′-Dichlorodihydrofluorescein diacetate (DCFDA). Radiation significantly increased ROS generation in HuR knockdown cells compared to control cells. To further test the involvement of ROS in radiosensitivity, control and HuR-silenced cells were pre-treated with N-Acetyl-L- cysteine (NAC), an ROS scavenger, prior to radiation. The presence of NAC completely prevented radiation sensitivity and ROS production, indicating the involvement of ROS in HuR-mediated radiation sensitivity. Second, we directly tested the involvement of the DNA damage response (DDR) pathway in radiosensitivity after silencing HuR by evaluating the number of γ-H2AX foci (a common indicator of DNA damage) in control and HuR-silenced cells following irradiation. Our results showed that the number of γ-H2AX foci was significantly greater in HuR-silenced cells than in control cells at 1 h, 2 h and 24 h after irradiation. The persistence of γ-H2AX foci suggests that radiosensitization by HuR silencing involves inhibition of the repair of damaged DNA. This hypothesis was supported by the comet assay, which showed that HuR-silenced cells had larger and longer-lasting tails than control cells, in keeping with the higher levels of DNA damage seen after silencing HuR. Our studies indicate that radiosensitization upon HuR knockdown is linked to suppression of the cellular response to genotoxic and oxidative damage. Citation Format: Meghna Mehta, James Griffith, Kanthesh Basalingappa, Anish Babu, Narsireddy Amreddy, Ranganayaki Muralidharan, Myriam Gorospe, Terence Herman, Wei-Qun Ding, Rajagopal Ramesh, Anupama Munshi. The RNA-binding protein HuR radiosensitizes human TNBC cells by modulating the cellular response to DNA damage and oxidative stress. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3306. doi:10.1158/1538-7445.AM2015-3306