Sirna-Mediated Hur Silencing Sensitizes Triple-Negative Breast Cancer Cells To Radiation Therapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA HuR is a ubiquitously expressed member of the Elav/Hu family of RNA-binding proteins which can associate with mRNAs containing AU-rich elements in their 3′-untranslated regions. It is predominantly a nuclear protein that translocates to the cytoplasm in response to stress signals and stabilizes mRNAs encoding proteins implicated in cell proliferation, angiogenesis, apoptosis, and stress response. Studies examining HuR expression in human cancers indicated that elevated cytoplasmic HuR expression is associated with a high histologic grade, large tumor size, and poor survival of patients with cancer, leading to the hypothesis that cytoplasmic HuR abundance could be a prognostic marker in cancer patients. It has been reported that altering the subcellular distribution of HuR leads to a decrease in mRNA stability and increases tamoxifen responsiveness in breast cancer cells. As the role of HuR in radiation resistance has not been previously evaluated, we designed this study to determine the role of HuR in mediating radiation response of human breast cancer cells. Subcellular fractionation studies in a panel of breast cancer cells [triple negative (MDA-MB-231, MDA-MB-468 and Hs578t), luminal (MCF-7), and normal mammary epithelial (MCF-10a)] demonstrated elevated cytoplasmic levels of HuR in the more aggressive triple negative breast cancer cells (TNBC) compared to the normal mammary and the luminal cells. TNBCs also had high expression of HuR mRNA and total protein as observed by quantitative (Q) RT-PCR and western blot analysis. To test if high expression of HuR contributed to the radiation resistance of TNBCs, HuR was silenced and cells were exposed to various doses of radiation. Clonogenic assays indicated that silencing HuR enhanced tumor cell radiosensitivity in MDA-MB-231, MDA-MB-468 and Hs578t cells, with the survival fraction at 2Gy declining from 59%, 49%, 65% in control (scrambled siRNA-transfected cells) to 40%, 33%, 46% in HuR-silenced cells, respectively. MCF-7 and MCF-10a cells were not radiosensitized upon HuR silencing. Since HuR plays a central role in cancer it is possible that multiple pathways and mechanisms are affected by HuR knockdown and could contribute to the observed radiosensitivity. Molecular studies suggested that HuR silencing in combination with radiation modulated the expression of several genes involved in cell survival, cell cycle and DNA repair in MDA-MB-231 cells. The involvement of the DNA repair pathway following treatment with siHuR was assessed using γ-H2AX foci as a marker. Our results show that a higher number of radiation-induced γ-H2AX foci are present in HuR-silenced MDA-MB-231 cells compared with control cells, suggesting a suppression of the double-strand DNA repair pathway. The persistence of γ-H2AX foci was not seen in the MCF-7 cells. We propose that HuR knockdown enhances the radioresponse of TNBC cells by inhibiting the repair of radiation-induced double-strand DNA breaks. Citation Format: Kanthesh M. Basalingappa, Meghna Mehta, James N. Griffith, Ranganayaki Muralidharan, Myriam Gorospe, Rajagopal Ramesh, Anupama Munshi. siRNA-mediated HuR silencing sensitizes triple-negative breast cancer cells to radiation therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3943. doi:10.1158/1538-7445.AM2014-3943