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.

Keywords: DNA repair; HuR; breast cancer; radiation; siRNA.

Figure 1. HuR is overexpressed in TNBC cells

A. Analysis of whole cell extracts for HuR by western blotting showed HuR expression was higher in TNBC cell lines compared to normal mammary epithelial cells. Actin was used as loading control. B. RNA was extracted from breast cancer cell lines and relative expression of HuR mRNA was assessed by quantitative RT-PCR analysis (normalized against GAPDH). Data are expressed as means ± SE of minimum three independent experiments. Asterisk denotes significance (p ≤ 0.05).

Figure 2. Effect of HuR silencing on the expression of HuR protein and mRNA

A. siHuR- treated TNBC cells showed reduced HuR protein expression with concomitant increase in p27 expression compared to siScr-treated cells. Actin was used as a loading control. B. HuR mRNA was significantly downregulated in siHuR-treated TNBC cell lines compared to siScr-treated cells. Asterisk denotes significance (p ≤ 0.05).

Figure 3. HuR silencing radiosensitizes human triple negative breast cancer cells

MDA-MB-468, MDA-MB-231 and Hs578t cells transfected with siHuR showed significant radiosensitization compared to siScr-transfected cells. Data represent the average of three independent experiments each plated in triplicate: solid line, siScr; dotted line, siHuR. Error bars represent ±SE (*p ≤ 0.05).

Figure 4. Modulation of HuR targets and DNA repair proteins upon siHuR and radiation treatment

MDA-MB-231 cells transfected with siScr or siHuR were irradiated and harvested 2 hours later. A. Western blot analysis showing suppression of HuR target proteins. B. RT-qPCR analysis of HuR and HuR-target mRNAs. C. Suppression of DNA repair proteins after siHuR plus radiation treatment compared to siScr plus radiation treatment.

Figure 5. HuR depletion prolongs γ-H2AX expression

Sub-confluent MDA-MB-231 cells were transiently transfected with siScr or siHuR (100 nM for 24 h) followed by a single 2 Gy dose of radiation. Samples were then incubated for various times after irradiation and stained for γ-H2AX foci A. Representative photomicrographs of MDA-MB-231 from various treatment conditions and time points from three independent experiments are shown. Blue stain: DAPI (nuclei). Green stain: γ-H2AX foci. B. γ-H2AX foci were quantified and plotted as the number of foci per nucleus. Mean ± SE number of foci per nucleus is shown. Asterisk denotes significance (p ≤ 0.05).

Figure 6. Silencing HuR enhances radiation induced DSBs

MDA-MB-231 cells were transfected with either siScr or siHuR and 24 hours later were irradiated at 20 Gy and harvested at the indicated times. A. Representative comet images of DSBs detected by neutral comet assay demonstrate the kinetics of tail moment in siScr or siHuR transfected cells at 0, 1 and 24 hours after irradiation. B. Distribution of DNA damage in cells treated as described in panel A. Olive tail moment (OTM) values were determined following the algorithm (olive tail moment = tail mean - head mean) tail% DNA/100) using Casplab software. Error bars represent SE. Asterisk denotes significance (p ≤ 0.05).

Figure 7. HuR knockdown inhibits thioredoxin reductase and enhances radiation-induced ROS production

A. MDA-MB-231 cells transfected with siScR or siHuR were incubated with 20 μM of H2DCFDA for 1 hour at 37°C. The cells were then irradiated at 8 Gy, harvested, and fluorescence activity measured at 495 nm excitation and 529 nm emission. Increase in ROS production was observed in siHuR-treated cells compared to siScr. ROS production was significantly increased when combined with radiation in both siScr and siHuR-treated cells. “NS” denotes no significance. B. Thioredoxin reductase (TrxR) activity was reduced in siHuR-treated MDA-MB-231 cells both in the presence and absence of radiation compared to siScr-treated cells. Asterisk denotes significance (p ≤ 0.05). C. siScr- and siHuR-treated cells were treated or treated for 1 hour with 10 μM NAC prior to radiation. The cells were subsequently radiated at 2, 4 and 6 Gy and subjected to clonogenic cell survival assay. siHuR-mediated radiosensitization was significantly abrogated in NAC-pretreated cells. Asterisk denotes significance (p ≤ 0.05).