HuR is an mRNA-binding protein whose overexpression in cancer cells has been associated with poor prognosis and resistance to therapy. While reports on HuR overexpression contributing to chemoresistance exist, limited information is available on HuR and radioresistance especially in triple-negative breast cancer (TNBC).In this study we investigated the role of HuR in radiation resistance in three TNBC (MDA-MB-231, MDA-MB-468 and Hs578t) cell lines. Endogenous HuR expression was higher in TNBC cells compared to normal cells. siRNA mediated knockdown of HuR (siHuR) markedly reduced HuR mRNA and protein levels compared to scrambled siRNA (siScr) treatment. Further, siHuR treatment sensitized TNBC cells to ionizing radiation at 2 Gy compared to siScr treatment as evidenced by the significant reduction in clonogenic cell survival from 59%, 49%, and 65% in siScr-treated cells to 40%, 33%, and 46% in siHuR-treated MDA-MB-231, MDA-MB-468 and Hs578t cells, respectively. Molecular studies showed increased ROS production and inhibition of thioredoxin reductase (TrxR) in HuR knockdown cells contributed to radiosensitization. Associated with increased ROS production was evidence of increased DNA damage, demonstrated by a significant increase (p < 0.05) in γ-H2AX foci that persisted for up to 24 h in siHuR plus radiation treated cells compared to control cells. Further, comet assay revealed that HuR-silenced cells had larger and longer-lasting tails than control cells, indicating higher levels of DNA damage. In conclusion, our studies demonstrate that HuR knockdown in TNBC cells elicits oxidative stress and DNA damage resulting in radiosensitization.

The Hippo pathway is an evolutionarily conserved signaling pathway that regulates proliferation and apoptosis to control organ size during developmental growth. Yes-associated protein 1 (YAP1), the terminal effector of the Hippo pathway, is a transcriptional co-activator and a potent growth promoter that has emerged as a critical oncogene. Overexpression of YAP1 has been implicated in promoting resistance to chemo-, radiation and targeted therapy in various cancers. However, the role of YAP1 in radioresistance in triple-negative breast cancer (TNBC) is currently unknown. We evaluated the role of YAP1 in radioresistance in TNBC in vitro, using two approaches to inhibit YAP1: 1) genetic inhibition by YAP1 specific shRNA or siRNA, and 2) pharmacological inhibition by using the small molecule inhibitor, verteporfin that prevents YAP1 transcriptional activity. Our findings demonstrate that both genetic and pharmacological inhibition of YAP1 sensitizes TNBC cells to radiation by inhibiting the EGFR/PI3K/AKT signaling axis and causing an increased accumulation of DNA damage. Our results reveal that YAP1 activation exerts a protective role for TNBC cells in radiotherapy and represents a pharmacological target to enhance the anti-tumor effects of DNA damaging modalities in the treatment of TNBC.

Tumor suppressor ARID1A, a subunit of the chromatin remodeling complex SWI/SNF, regulates cell cycle progression, interacts with the tumor suppressor TP53, and prevents genomic instability. In addition, ARID1A has been shown to foster resistance to cancer therapy. By promoting non-homologous end joining (NHEJ), ARID1A enhances DNA repair. Consequently, ARID1A has been proposed as a promising therapeutic target to sensitize cancer cells to chemotherapy and radiation. Here, we report that ARID1A is regulated by human antigen R (HuR), an RNA-binding protein that is highly expressed in a wide range of cancers and enables resistance to chemotherapy and radiation. Our results indicate that HuR binds ARID1A mRNA, thereby increasing its stability in breast cancer cells. We further find that ARID1A expression suppresses the accumulation of DNA double-strand breaks (DSBs) caused by radiation and can rescue the loss of radioresistance triggered by HuR inhibition, suggesting that ARID1A plays an important role in HuR-driven resistance to radiation. Taken together, our work shows that HuR and ARID1A form an important regulatory axis in radiation resistance that can be targeted to improve radiotherapy in breast cancer patients.

No abstract

Overexpression of BMI1 in human cancer cells, a member of the polycomb group of repressive complexes, correlates with advanced stage of disease, aggressive clinico-pathological behavior, poor prognosis, and resistance to radiation and chemotherapy. Studies have shown that experimental reduction of BMI1 protein level in tumor cells results in inhibition of cell proliferation, induction of apoptosis and/or senescence, and increased susceptibility to cytotoxic agents and radiation therapy. Although a role for BMI1 in cancer progression and its importance as a molecular target for cancer therapy has been established, information on the impact of silencing BMI1 in triple-negative breast cancer (TNBC) and its consequence on radiotherapy have not been well studied. Therefore, in the present study we investigated the potential therapeutic benefit of radiation therapy in BMI1-silenced breast cancer cells and studied the mechanism(s) of radiosensitization. Human MDA-MB-231 and SUM159PT breast cancer cells that were either stably transfected with a lentiviral vector expressing BMI1 shRNA (shBMI1) or control shRNA (shControl) or transient transfection with a BMI1-specific siRNA were used. Silencing of BMI1 resulted in marked reduction in BMI1 both at the mRNA and protein level that was accompanied by a significant reduction in cell migration compared to control cells. Further, BMI1 knockdown produced a marked enhancement of DNA damage as evidenced by Comet Assay and γH2AX foci, resulting in a dose-dependent radiosensitization effect. Molecular studies revealed modulation of protein expression that is associated with the DNA damage response (DDR) and autophagy pathways. Our results demonstrate that BMI1 is an important therapeutic target in breast cancer and suppression of BMI1 produces radiation sensitivity. Further, combining BMI1-targeted therapeutics with radiation might benefit patients diagnosed with TNBC.

Purpose/Objective(s): Radiation-induced neurogenesis impairment in hippocampus was considered to play a key role in cognitive deficits arise from exposure to ionizing radiation, but the mechanism remains elusive. This study aimed to investigate the role of TrkA、TrkB in radiationinduced hippocampal neurogenesis impairment. Materials/Methods: A singal dose of 10Gy 4MV electron beam were given to young male Sprague-Dawley rats (50e60g, day 21). Western blot was performed and the brain sections were immunohistochemical stained. Q-PCR were applied to detect mRNA levels.The alterations of TrkA/TrkB protein and mRNA levels and hippocampal neurogenesis were observed and analyzed. Golgi staining was used to observe the dendritic spine of hippocampus.To evaluate the consequence of whole brain irradiation on hippocampus-dependent memory formation, Morris water maze test, Novel object recognition and open field test were performed. Results: The results show that compared with control group, the numbers of dendritic spine significantly decreased after irradiation and its shape change obviously. Immunofluorescence showed a significant decrease in neural precursors prolifration comparing with control group (53%). Protein levels of TrkA expressions increased significantly (32%) while the levels of TrkB expressions decreased significantly (43%); the mRNA levels of TrkA expressions increased significantly (27%) while the levels of TrkB expressions decreased significantly (36%). In Morris water maze test, passive-avoidance test and open field test, we confirmed whole brain irradiation lead to notable memory impairment at day 30 after irradiation. Conclusion: As a signaling pathways downstream of NGF and BDNF, TrkA,TrkB may play an important role in radiation-induced neurogenesis impairment. Future studies are necessary to determine the strong association between TrkA and TrkB after whole brain irradiation.

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

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