Silencing BMI1 radiosensitizes human breast cancer cells by inducing DNA damage and autophagy

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.

Figure 1.

BMI1 knockdown evaluated by (A) western blotting and (B) quantitative RT-PCR in MDA-MB-231 cells. (C) BMI1 expression in siBMI1-treated SUM159PT cells. (D) wound healing assay in MDA-MB-231 cells showed shBMI1 reduced cell migration and (E) tumor cell migration of MDA-MB-231 cells was significantly inhibited by silencing BMI1. Error bars represent standard error of the mean.

Figure 2.

Silencing BMI1 radiosensitizes TNBC cells. (A) MDA-MB-231 shControl or shBMI1 cells and (B) SUM159PT cells treated with siScr or siBMI1 were assessed for radiosensitization by clonogenic cell survival assay immediately after irradiation. BMI1 silencing radiosensitized both cell lines compared to controls. The results represent the average of three independent experiments, each plated in triplicate. Error bars represent standard error of the mean.

Figure 3.

Silencing BMI1 induces DNA damage in MDA-MB-231 cells. Sub-confluent MDA-MB-231 shControl or shBMI1 cells were grown on coverslips for 24 h, exposed to 2 Gy dose of radiation, incubated for various times after irradiation and stained for γ-H2AX. (A) Representative photomicrographs of MDA-MB-231 from various treatment conditions and time points are shown. 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 are shown. *p≤0.05, **p<0.005. (C) Western blotting for γ-H2AX showed increased γ-H2AX expression in shBMI1 plus radiation treated MDA-MB-231 cells compared to controls. β-actin was used as a loading control.

Figure 4.

Targeting BMI1 causes DNA damage. (A) Representative Comet images of DSBs detected by neutral Comet assay demonstrate the kinetics of tail moment in shControl or shBMI1 cells without radiation or 24 h post-irradiation. (B) Increased comet tail moment was observed in shBMI1 cells compared to shControl at 0 and 24 h post-irradiation. 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 standard error of the mean. **p≤0.005.

Figure 5.

BMI1 modulates apoptotic and DNA damage response protein expression. (A and C) MDA-MB-231 (shControl and shBMI1) and (B) SUM159PT (siScr and siBMI1) cells were grown for 24 h and treated with or without 5 Gy radiation. Two hours post-radiation, cell lysates were evaluated by western blotting using the respective antibodies. β-actin was used as a loading control.

Figure 6.

BMI1 influences autophagy. Immunoblot analysis of autophagy proteins in (A) MDA-MB-231 shControl and shBMI1; (B) SUM159PT siScr and siBMI1 cells grown for 24 h and treated with or without 5 Gy radiation for 24 and 48 h. β-actin was used as a loading control. (C) qRT-PCR analysis for autophagy markers in MDA-MB-231 shControl or shBMI1 cells grown for 24 h and treated with or without 5 Gy radiation for 2 h, normalized to GAPDH. Results showed an increase in autophagy markers in shBMI1 and shBMI1 plus radiation treated cells compared to controls.