A greater tail length and a greater tail area indicate that more DNA breaks have occurred. 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 H2AX 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 malignancy and suppression of BMI1 produces radiation sensitivity. Further, combining BMI1-targeted therapeutics with radiation might benefit patients diagnosed with TNBC. Keywords: autophagy, BMI1, breast cancer, radiation, DNA damage Introduction The polycomb group (PcG) of transcription factor proteins form transcriptional repressor modules that play crucial roles in many physiological processes, including cell differentiation, stem cell self-renewal, and gene silencing through histone modifications (1). Numerous studies have shown that PcG proteins are involved in malignant transformation and tumor development in various malignancy types (2). B cell-specific Moloney murine leukemia computer virus integration site 1 (BMI1), a member of the PcG complex, plays an essential role in the maintenance and self-renewal of hematopoietic and neural stem cells, at least partly by silencing the Ink4a/Arf locus (3,4). BMI1 has also been linked with a multitude of cellular processes, including cell cycle progression, apoptosis, epithelial-to-mesenchymal transition (EMT), senescence, TRV130 HCl (Oliceridine) immortalization and/or induction of telomerase (5C7). BMI1 overexpression is usually associated with disease progression and poor clinical outcome in a number of human malignancies (8C11). Although BMI1 plays a critical role in cancer, the precise molecular mechanism by which it contributes to malignancy development and therapy failure remains poorly comprehended. Several impartial studies have exhibited that genetic silencing and pharmacologic inhibition of BMI1 suppresses the growth of TRV130 HCl (Oliceridine) various cancers, induces cell cycle arrest, apoptosis and senescence, and increases susceptibility to chemotherapeutic brokers and ionizing radiation (12C14). In normal human keratinocytes, BMI1 elicits radioprotective effects by mitigating TRV130 HCl (Oliceridine) the genotoxic effects of ionizing radiation (IR) (15). In nasopharyngeal carcinoma cells, targeting BMI1 expression increases their susceptibility to radiation through the induction of oxidative stress and apoptosis (13). Elevated expression of BMI1 has been shown to radioprotect CD133-positive cancer-initiating neural stem cells through recruitment of DNA damage response (DDR) machinery to DSBs after exposure to radiation (16). Although a role for BMI1 in cancer progression and its importance as a target for therapy has been reported, its role in radiosensitization of breast cancer has not been investigated. In the present study, we demonstrate that silencing BMI1 sensitizes MDA-MB-231 and SUM159PT breast malignancy cells to ionizing radiation. We also show that this sensitization occurs through induction of both the DDR and autophagy pathways. These results indicate that BMI1 may play an important role in radioresistance, and that BMI1 suppression may be an important therapeutic target for breast malignancy. Materials and methods Cell lines Human MDA-MB-231 breast malignancy cell line obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) was maintained in -MEM (Cellgro, Manassas, VA, USA) made up of 10% fetal bovine serum, 2 mmol/l L-glutamine, and 2 mmol/l penicillin-streptomycin. SUM159PT cells were obtained from Asterand Bioscience (Detroit, MI, USA) and maintained in Ham’s F-12 media supplemented with 5% heat-inactivated FBS, 2 mmol/l penicillin-streptomycin, 10 mM Hepes, and 1 g/ml insulin. All cultures were maintained at 37C in an atmosphere of 5% CO2 and 95% room air. Plasmid construction Sequences (miR shControl: Sense 5-AGCGATCTCGCTTGGGCGAGAGTAAGTATGAAGCCACAGATGTGACTTACTCTCGCCCAACGAGAG-3, Antisense 5-GGCAACTCTCGCTTGGGCGAGAGTAAGTACATCTGTGGCTTCACTACTTACTCTCGCCCAAGCGAGAT-3; miR shBMI1: Sense 5-AGCGATCCAAGATATTGTATACAAATTAGTGAAGCCACAGATGTAATTTGTATACAATATCTTGGAG-3, Antisense 5-GGCACTCCAAGATATTGTATACAAATTACATCTGTGGCTTCACTAATTTGTATACAATATCTTGGAT-3) were cloned into pEN_RmiRc2 (17). Then, entry vectors made up of shRNA sequence were recombined with the lentiviral destination vector CMV PURO DEST according to manufacturer’s recommendations (Invitrogen, Grand Island, NY, USA). siRNA transfection Transfection of SUM159PT cells with human BMI1 siRNA (siBMI1) and non-targeting siRNA#3 (siScr) (GE Dharmacon, Lafayette, CO, USA) was performed in 60-mm dishes using DharmaFECT 2 transfection reagent (GE Dharmacon) according to manufacturer’s instructions. Cells were transfected with siRNA (20 nM) in serum-free medium. Six hours after transfection, the media was replaced with fresh medium made up of 2% serum. The next day the Rabbit polyclonal to AACS cells were irradiated (5 Gy) and harvested after specified incubation.