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

Griffith,James; Andrade,Daniel; Mehta,Meghna; Berry,William; Benbrook,Doris  M.; Aravindan,Natarajan; Herman,Terence S.; Ramesh,Rajagopal; Munshi,Anupama

doi:10.3892/or.2017.5478

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

Authors:
- James Griffith
- Daniel Andrade
- Meghna Mehta
- William Berry
- Doris M. Benbrook
- Natarajan Aravindan
- Terence S. Herman
- Rajagopal Ramesh
- Anupama Munshi
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Affiliations: Department of Radiation Oncology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA, Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA, Department of Obstetrics and Gynecology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA, Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
Published online on: February 28, 2017 https://doi.org/10.3892/or.2017.5478
Pages: 2382-2390

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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.

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1	Simon JA and Kingston RE: Mechanisms of polycomb gene silencing: Knowns and unknowns. Nat Rev Mol Cell Biol. 10:697–708. 2009.PubMed/NCBI
2	Wang W, Qin JJ, Voruganti S, Nag S, Zhou J and Zhang R: Polycomb group (PcG) proteins and human cancers: Multifaceted functions and therapeutic implications. Med Res Rev. 35:1220–1267. 2015. View Article : Google Scholar : PubMed/NCBI
3	Park IK, Qian D, Kiel M, Becker MW, Pihalja M, Weissman IL, Morrison SJ and Clarke MF: Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature. 423:302–305. 2003. View Article : Google Scholar : PubMed/NCBI
4	Bruggeman SW, Valk-Lingbeek ME, van der Stoop PP, Jacobs JJ, Kieboom K, Tanger E, Hulsman D, Leung C, Arsenijevic Y, Marino S, et al: Ink4a and Arf differentially affect cell proliferation and neural stem cell self-renewal in Bmi1-deficient mice. Genes Dev. 19:1438–1443. 2005. View Article : Google Scholar : PubMed/NCBI
5	Yang MH, Hsu DS, Wang HW, Wang HJ, Lan HY, Yang WH, Huang CH, Kao SY, Tzeng CH, Tai SK, et al: Bmi1 is essential in Twist1-induced epithelial-mesenchymal transition. Nat Cell Biol. 12:982–992. 2010. View Article : Google Scholar : PubMed/NCBI
6	Jacobs JJ, Kieboom K, Marino S, DePinho RA and van Lohuizen M: The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus. Nature. 397:164–168. 1999. View Article : Google Scholar : PubMed/NCBI
7	Zhang FB, Sui LH and Xin T: Correlation of Bmi-1 expression and telomerase activity in human ovarian cancer. Br J Biomed Sci. 65:172–177. 2008. View Article : Google Scholar : PubMed/NCBI
8	Vonlanthen S, Heighway J, Altermatt HJ, Gugger M, Kappeler A, Borner MM, van Lohuizen M and Betticher DC: The bmi-1 oncoprotein is differentially expressed in non-small cell lung cancer and correlates with INK4A-ARF locus expression. Br J Cancer. 84:1372–1376. 2001. View Article : Google Scholar : PubMed/NCBI
9	Kim JH, Yoon SY, Kim CN, Joo JH, Moon SK, Choe IS, Choe YK and Kim JW: The Bmi-1 oncoprotein is overexpressed in human colorectal cancer and correlates with the reduced p16^INK4a/p14^ARF proteins. Cancer Lett. 203:217–224. 2004. View Article : Google Scholar : PubMed/NCBI
10	Kim JH, Yoon SY, Jeong SH, Kim SY, Moon SK, Joo JH, Lee Y, Choe IS and Kim JW: Overexpression of Bmi-1 oncoprotein correlates with axillary lymph node metastases in invasive ductal breast cancer. Breast. 13:383–388. 2004. View Article : Google Scholar : PubMed/NCBI
11	Silva J, García V, García JM, Peña C, Domínguez G, Díaz R, Lorenzo Y, Hurtado A, Sánchez A and Bonilla F: Circulating Bmi-1 mRNA as a possible prognostic factor for advanced breast cancer patients. Breast Cancer Res. 9:R552007. View Article : Google Scholar : PubMed/NCBI
12	Zhang R, Xu LB, Yue XJ, Yu XH, Wang J and Liu C: Bmi1 gene silencing inhibits the proliferation and invasiveness of human hepatocellular carcinoma cells and increases their sensitivity to 5-fluorouracil. Oncol Rep. 29:967–974. 2013.PubMed/NCBI
13	Xu XH, Liu XY, Su J, Li DJ, Huang Q, Lu MQ, Yi F, Ren JH and Chen WH: ShRNA targeting Bmi-1 sensitizes CD44⁺ nasopharyngeal cancer stem-like cells to radiotherapy. Oncol Rep. 32:764–770. 2014.PubMed/NCBI
14	Wang G, Liu L, Sharma S, Liu H, Yang W, Sun X and Dong Q: Bmi-1 confers adaptive radioresistance to KYSE-150R esophageal carcinoma cells. Biochem Biophys Res Commun. 425:309–314. 2012. View Article : Google Scholar : PubMed/NCBI
15	Dong Q, Oh JE, Chen W, Kim R, Kim RH, Shin KH, McBride WH, Park NH and Kang MK: Radioprotective effects of Bmi-1 involve epigenetic silencing of oxidase genes and enhanced DNA repair in normal human keratinocytes. J Invest Dermatol. 131:1216–1225. 2011. View Article : Google Scholar : PubMed/NCBI
16	Facchino S, Abdouh M, Chatoo W and Bernier G: BMI1 confers radioresistance to normal and cancerous neural stem cells through recruitment of the DNA damage response machinery. J Neurosci. 30:10096–10111. 2010. View Article : Google Scholar : PubMed/NCBI
17	Long CL, Berry WL, Zhao Y, Sun XH and Humphrey MB: E proteins regulate osteoclast maturation and survival. J Bone Miner Res. 27:2476–2489. 2012. View Article : Google Scholar : PubMed/NCBI
18	Muralidharan R, Panneerselvam J, Chen A, Zhao YD, Munshi A and Ramesh R: HuR-targeted nanotherapy in combination with AMD3100 suppresses CXCR4 expression, cell growth, migration and invasion in lung cancer. Cancer Gene Ther. 22:581–590. 2015. View Article : Google Scholar : PubMed/NCBI
19	Munshi A, Tanaka T, Hobbs ML, Tucker SL, Richon VM and Meyn RE: Vorinostat, a histone deacetylase inhibitor, enhances the response of human tumor cells to ionizing radiation through prolongation of gamma-H2AX foci. Mol Cancer Ther. 5:1967–1974. 2006. View Article : Google Scholar : PubMed/NCBI
20	Munshi A, Kurland JF, Nishikawa T, Tanaka T, Hobbs ML, Tucker SL, Ismail S, Stevens C and Meyn RE: Histone deacetylase inhibitors radiosensitize human melanoma cells by suppressing DNA repair activity. Clin Cancer Res. 11:4912–4922. 2005. View Article : Google Scholar : PubMed/NCBI
21	Kroemer G, Mariño G and Levine B: Autophagy and the integrated stress response. Mol Cell. 40:280–293. 2010. View Article : Google Scholar : PubMed/NCBI
22	Proctor E, Waghray M, Lee CJ, Heidt DG, Yalamanchili M, Li C, Bednar F and Simeone DM: Bmi1 enhances tumorigenicity and cancer stem cell function in pancreatic adenocarcinoma. PLoS One. 8:e558202013. View Article : Google Scholar : PubMed/NCBI
23	Santivasi WL and Xia F: Ionizing radiation-induced DNA damage, response, and repair. Antioxid Redox Signal. 21:251–259. 2014. View Article : Google Scholar : PubMed/NCBI
24	Khanna KK and Jackson SP: DNA double-strand breaks: Signaling, repair and the cancer connection. Nat Genet. 27:247–254. 2001. View Article : Google Scholar : PubMed/NCBI
25	Olive PL: The role of DNA single- and double-strand breaks in cell killing by ionizing radiation. Radiat Res. 150:(Suppl). S42–S51. 1998. View Article : Google Scholar : PubMed/NCBI
26	Liu J, Cao L, Chen J, Song S, Lee IH, Quijano C, Liu H, Keyvanfar K, Chen H, Cao LY, et al: Bmi1 regulates mitochondrial function and the DNA damage response pathway. Nature. 459:387–392. 2009. View Article : Google Scholar : PubMed/NCBI
27	Apel A, Herr I, Schwarz H, Rodemann HP and Mayer A: Blocked autophagy sensitizes resistant carcinoma cells to radiation therapy. Cancer Res. 68:1485–1494. 2008. View Article : Google Scholar : PubMed/NCBI
28	Fujiwara K, Iwado E, Mills GB, Sawaya R, Kondo S and Kondo Y: Akt inhibitor shows anticancer and radiosensitizing effects in malignant glioma cells by inducing autophagy. Int J Oncol. 31:753–760. 2007.PubMed/NCBI
29	Sui X, Kong N, Ye L, Han W, Zhou J, Zhang Q, He C and Pan H: p38 and JNK MAPK pathways control the balance of apoptosis and autophagy in response to chemotherapeutic agents. Cancer Lett. 344:174–179. 2014. View Article : Google Scholar : PubMed/NCBI
30	Ge J, Liu Y, Li Q, Guo X, Gu L, Ma ZG and Zhu YP: Resveratrol induces apoptosis and autophagy in T-cell acute lymphoblastic leukemia cells by inhibiting Akt/mTOR and activating p38-MAPK. Biomed Environ Sci. 26:902–911. 2013.PubMed/NCBI
31	Dong Q, Sharma S, Liu H, Chen L, Gu B, Sun X and Wang G: HDAC inhibitors reverse acquired radio resistance of KYSE-150R esophageal carcinoma cells by modulating Bmi-1 expression. Toxicol Lett. 224:121–129. 2014. View Article : Google Scholar : PubMed/NCBI
32	Gong XM, Zhang Q, Torossian A, Cao JP and Fu S: Selective radiosensitization of human cervical cancer cells and normal cells by artemisinin through the abrogation of radiation-induced G2 block. Int J Gynecol Cancer. 22:718–724. 2012. View Article : Google Scholar : PubMed/NCBI
33	Wu J, Hu D, Yang G, Zhou J, Yang C, Gao Y and Zhu Z: Down-regulation of BMI-1 cooperates with artemisinin on growth inhibition of nasopharyngeal carcinoma cells. J Cell Biochem. 112:1938–1948. 2011. View Article : Google Scholar : PubMed/NCBI
34	Kreso A, van Galen P, Pedley NM, Lima-Fernandes E, Frelin C, Davis T, Cao L, Baiazitov R, Du W, Sydorenko N, et al: Self-renewal as a therapeutic target in human colorectal cancer. Nat Med. 20:29–36. 2014. View Article : Google Scholar : PubMed/NCBI
35	Bolomsky A, Schlangen K, Schreiner W, Zojer N and Ludwig H: Targeting of BMI-1 with PTC-209 shows potent anti-myeloma activity and impairs the tumour microenvironment. J Hematol Oncol. 9:172016. View Article : Google Scholar : PubMed/NCBI
36	Mayr C, Wagner A, Loeffelberger M, Bruckner D, Jakab M, Berr F, Di Fazio P, Ocker M, Neureiter D, Pichler M, et al: The BMI1 inhibitor PTC-209 is a potential compound to halt cellular growth in biliary tract cancer cells. Oncotarget. 7:745–758. 2016.PubMed/NCBI