title: HuR silencing elicits oxidative stress and DNA damage and sensitizes human triple-negative breast cancer cells to radiotherapy.
abstract: 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.
authors: Department of Radiation Oncology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors: Meghna
authors: M
authors: Mehta
authors: Department of Radiation Oncology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors: Kanthesh
authors: K
authors: Basalingappa
authors: Department of Radiation Oncology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors: James N
authors: JN
authors: Griffith
authors: Department of Radiation Oncology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors: Daniel
authors: D
authors: Andrade
authors: Department of Pathology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.|Stephenson Cancer Center, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors: Anish
authors: A
authors: Babu
authors: Department of Pathology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.|Stephenson Cancer Center, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors: Narsireddy
authors: N
authors: Amreddy
authors: Department of Pathology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.|Stephenson Cancer Center, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors: Ranganayaki
authors: R
authors: Muralidharan
authors: National Institute on Aging, National Institutes of Health, Baltimore, Maryland, USA.
authors: Myriam
authors: M
authors: Gorospe
authors: Department of Radiation Oncology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors: Terence
authors: T
authors: Herman
authors: Department of Pathology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors: Wei-Qun
authors: WQ
authors: Ding
authors: Department of Pathology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.|Stephenson Cancer Center, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.|Graduate Program in Biomedical Sciences, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors: Rajagopal
authors: R
authors: Ramesh
authors: Department of Radiation Oncology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.|Stephenson Cancer Center, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors: Anupama
authors: A
authors: Munshi
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism* / Q000532:radiotherapy; D017209:Apoptosis; D045744:Cell Line, Tumor; D020552:Comet Assay; D004249:DNA Damage / Q000235:genetics; D000067780:ELAV-Like Protein 1 / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D018384:Oxidative Stress / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D011836:Radiation Tolerance; D011839:Radiation, Ionizing; D017382:Reactive Oxygen Species / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D064726:Triple Negative Breast Neoplasms / Q000378:metabolism* / Q000532:radiotherapy
chemical_list: D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D013880:Thioredoxin-Disulfide Reductase
keywords: DNA repair; HuR; breast cancer; radiation; siRNA