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.

Nanosomes carrying doxorubicin exhibit potent anticancer activity against human lung cancer cells

“…Total cells lysates prepared from lung tumor and normal cells receiving various treatments were subjected to Western blotting as previously described32 and is briefly illustrated in supplemetry section 3.…”

“…Double strandbreaks (DSBs) induced by free-Dox, NanoDox and nanosomes were determined by Comet assay (CometAssay ® kit; Trevigen, Inc., Gaithersburg, MD) as previously described32. The detail of the comet assay is outlined in supplementary section 4.…”

Nanoparticles for siRNA-Based Gene Silencing in Tumor Therapy

“…As cancers present with ever-increasing heterogeneity, siRNA-based therapy is increasingly being explored in combination with chemo- [127], [128], immune- [129]–[131], radiation- [132]–[134], and photodynamic therapies [135]–[137]. While gene silencing using siRNA improves the sensitivity of drug resistant cancer to chemotherapeutics [127] siRNA mediated downregulation of immunosuppressive genes enhances the sensitization of cancer cells towards T cell-mediated killing [129].…”

“…While gene silencing using siRNA improves the sensitivity of drug resistant cancer to chemotherapeutics [127] siRNA mediated downregulation of immunosuppressive genes enhances the sensitization of cancer cells towards T cell-mediated killing [129]. Reports also suggest that siRNA based silencing of specific genes enhances radiosensitization of cancer cells [133], [134]. In contrast, downregulation of certain genes protect normal tissues from harmful effect of radiation [132].…”

Tumor-targeted Nanoparticle Delivery of HuR siRNA Inhibits Lung Tumor Growth In Vitro and In Vivo By Disrupting the Oncogenic Activity of the RNA-binding Protein HuR

“…Recent studies from our laboratory demonstrated that HuR downregulation inhibited cell growth in non-small cell lung cancer (NSCLC) in vitro and altered the cells’ ability to undergo migration and invasion [12,15]. Further, we recently showed HuR inhibition radiosensitized human breast cancer cells [16]. All of these studies indicate HuR is a molecular target for cancer therapy and advancing preclinical testing of HuR-targeted therapies could ultimately lead to clinical testing.…”