title: Bmi1 regulates self-renewal and epithelial to mesenchymal transition in breast cancer cells through Nanog.
abstract: BACKGROUND
  The Bmi1 polycomb ring finger oncogene, a transcriptional repressor belonging to the Polycomb group of proteins plays an important role in the regulation of stem cell self-renewal and is elevated in several cancers. In the current study, we have explored the role of Bmi1 in regulating the stemness and drug resistance of breast cancer cells.
  
  
  METHODS
  Using real time PCR and immunohistochemistry primary breast tissues were analyzed. Retro- and lentiviruses were utilized to overexpress and knockdown Bmi1, RT-PCR and Western blot was performed to evaluate mRNA and protein expression. Stemness properties were analyzed by flow cytometry and sphere-formation and tumor formation was determined by mouse xenograft experiments. Dual luciferase assay was employed to assess promoter activity and MTT assay was used to analyze drug response.
  
  
  RESULTS
  We found Bmi1 overexpression in 64% of grade III invasive ductal breast adenocarcinomas compared to normal breast tissues. Bmi1 overexpression in immortalized and transformed breast epithelial cells increased their sphere-forming efficiency, induced epithelial to mesenchymal transition (EMT) with an increase in the expression of stemness-related genes. Knockdown of Bmi1 in tumorigenic breast cells induced epithelial morphology, reduced expression of stemness-related genes, decreased the IC50 values of doxorubicin and abrogated tumor-formation. Bmi1-high tumors showed elevated Nanog expression whereas the tumors with lower Bmi1 showed reduced Nanog levels. Overexpression of Bmi1 increased Nanog levels whereas knockdown of Bmi1 reduced its expression. Dual luciferase promoter-reporter assay revealed Bmi1 positively regulated the Nanog and NFκB promoter activity. RT-PCR analysis showed that Bmi1 overexpression activated the NFκB pathway whereas Bmi1 knockdown reduced the expression of NFκB target genes, suggesting that Bmi1 might regulate Nanog expression through the NFκB pathway.
  
  
  CONCLUSIONS
  Our study showed that Bmi1 is overexpressed in several high-grade, invasive ductal breast adenocarcinomas, thus supporting its role as a prognostic marker. While Bmi1 overexpression increased self-renewal and promoted EMT, its knockdown reversed EMT, reduced stemness, and rendered cells drug sensitive, thus highlighting a crucial role for Bmi1 in regulating the stemness and drug response of breast cancer cells. Bmi1 may control self-renewal through the regulation of Nanog expression via the NFκB pathway.
authors.forename: Anurag N
authors.initials: AN
authors.lastname: Paranjape
authors.forename: Sai A
authors.initials: SA
authors.lastname: Balaji
authors.forename: Tamoghna
authors.initials: T
authors.lastname: Mandal
authors.forename: Esthelin Vittal
authors.initials: EV
authors.lastname: Krushik
authors.forename: Pradeep
authors.initials: P
authors.lastname: Nagaraj
authors.forename: Geetashree
authors.initials: G
authors.lastname: Mukherjee
authors.affiliation: Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, Karnataka, India. anu@mrdg.iisc.ernet.in.
authors.forename: Annapoorni
authors.initials: A
authors.lastname: Rangarajan
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D055785:Gene Knockdown Techniques; D064593:Heterografts; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D000071317:Nanog Homeobox Protein; D060787:Neoplasm Grading; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D063150:Polycomb Repressive Complex 1 / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D047368:Tumor Burden / Q000187:drug effects / Q000235:genetics
chemical_list: D000903:Antibiotics, Antineoplastic; C069077:BMI1 protein, human; D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D004317:Doxorubicin; D063150:Polycomb Repressive Complex 1