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.
Highlights
Triple negative breast cancer (TNBC) is a distinct subset of breast cancer characterized by aggressive clinical behavior with limited treatment options and very poor prognosis
In this study we investigated whether Human antigen R (HuR) suppression could enhance the radiosensitivity of TNBC cells in vitro
We further tested the effects of silencing HuR on radiosensitization of TNBC cells and observed that all three TNBC lines were radiosensitized upon HuR knockdown (Figure 3)
Summary
Triple negative breast cancer (TNBC) is a distinct subset of breast cancer characterized by aggressive clinical behavior with limited treatment options and very poor prognosis. Unraveling the underlying mechanism controlling the development of resistance to radiation therapy can help delay or eliminate the development of resistance and pave the way for designing effective radiosensitizers. Ionizing radiation causes both direct and indirect damage to cells. Exposure to clinically relevant doses of ionizing radiation elicits genotoxic stress by triggering increased levels of ROS which creates oxidative stress and disturbs the redox balance within the cells leading to generation of DNA damage [6, 7]. Thioredoxin reductase (TrxR) is reported to be overexpressed in many aggressive cancers and plays a crucial role in redox balance and antioxidant function, including defense of oxidative stress [11,12]. Agents that can inhibit TrxR have the potential to be developed as novel anticancer agents and radiosensitizers [13, 14]
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