Bmi1 depletion enhances radiation sensitivity of tumour cells through induction of the p53-mediated apoptotic pathway

Introduction: In recent years, the polycomb group (PcG) gene family has emerged as key regulators of cancer development and progression. Several studies have correlated over-expression of different members of the PcG gene family, such as BMI1 and EZH2, with cancer development, progression, and therapy failure. However, the exact molecular mechanism(s) by which PcG genes mediate these processes remains to be fully elucidated. The purpose of this study was to assess the expression of BMI1 protein in primary NPC tissues and to investigate the role of BMI1 in the response of tumour cells to ionizing radiation therapy (RT).
 Methods: Quantitative RT-PCR and immunohistochemistry were used to assess BMI1 expression in snap-frozen and FFPE primary NPC specimens, respectively. Dharmacon SMARTpool siRNA was used to knockdown BMI1 expression in C666-1 NPC cells. The affymetrix gene expression array (HG-U133 plus 2.0) was utilized to measure changes in mRNA transcripts following BMI1-depletion + RT. Transmission Electron Microscopy (TEM) was utilized to visualize cellular changes (e.g. nuclear and mitochondrial condensation, autophagy) in response to treatment. Cell cycle analysis and caspase3-7 activation were utilized to measure apoptosis in vitro. MTT assay was used to assess cell viability in vitro. In vivo therapeutic experiments were conducted using SCID mice.
 Results: Our qRT-PCR data demonstrated over-expression (>2-fold) of BMI1 in 70% of primary NPC specimens and 100% of NPC xenograft tumours, which was further validated using immunohistochemistry. Interestingly, we also demonstrate a novel radioresistance function for BMI1 in NPC, whereby BMI1 depletion significantly increased the sensitivity of NPC cells to RT, which upon further analysis was determined to be a synergistic interaction. Genome-wide microarray analysis performed on cells depleted of BMI1 and exposed to RT revealed the induction of several TP53 target genes with pro-apototic function. Additional pathways induced by BMI1 depletion and RT include genes encoding for DNA repair, lysosomal, and cell surface proteins. Concordantly, BMI1 depletion combined with 6 Gy RT completely abrogated tumour formation capacity in vivo, demonstrating the potential therapeutic efficacy of this combinatorial strategy.
 Conclusions: Thus, our data are the first to demonstrate that BMI1 is conferring radioresistance in tumour cells through complex mechanisms, including down-regulation of p53-mediated apoptosis and provide a biological explanation for BMI1 over-expression conferring reduced survival in NPC patients. In addition, our data suggest a possible role for BMI1 in tumour development and progression through repression of DNA repair genes leading to genetic instability.