title: Generation of tumor-initiating cells by exogenous delivery of OCT4 transcription factor.
abstract: BACKGROUND
  Tumor-initiating cells (TIC) are being extensively studied for their role in tumor etiology, maintenance and resistance to treatment. The isolation of TICs has been limited by the scarcity of this population in the tissue of origin and because the molecular signatures that characterize these cells are not well understood. Herein, we describe the generation of TIC-like cell lines by ectopic expression of the OCT4 transcription factor (TF) in primary breast cell preparations.
  
  
  METHODS
  OCT4 cDNA was over-expressed in four different primary human mammary epithelial (HMEC) breast cell preparations from reduction mammoplasty donors. OCT4-transduced breast cells (OTBCs) generated colonies (frequency ~0.01%) in self-renewal conditions (feeder cultures in human embryonic stem cell media). Differentiation assays, immunofluorescence, immunohistochemistry, and flow cytometry were performed to investigate the cell of origin of OTBCs. Serial dilutions of OTBCs were injected in nude mice to address their tumorigenic capabilities. Gene expression microarrays were performed in OTBCs, and the role of downstream targets of OCT4 in maintaining self-renewal was investigated by knock-down experiments.
  
  
  RESULTS
  OTBCs overcame senescence, overexpressed telomerase, and down-regulated p16INK4A. In differentiation conditions, OTBCs generated populations of both myoepithelial and luminal cells at low frequency, suggesting that the cell of origin of some OTBCs was a bi-potent stem cell. Injection of OTBCs in nude mice generated poorly differentiated breast carcinomas with colonization capabilities. Gene expression microarrays of OTBC lines revealed a gene signature that was over-represented in the claudin-low molecular subtype of breast cancer. Lastly, siRNA-mediated knockdown of OCT4 or downstream embryonic targets of OCT4, such as NANOG and ZIC1, suppressed the ability of OTBCs to self-renew.
  
  
  CONCLUSIONS
  Transduction of OCT4 in normal breast preparations led to the generation of cell lines possessing tumor-initiating and colonization capabilities. These cells developed high-grade, poorly differentiated breast carcinomas in nude mice. Genome-wide analysis of OTBCs outlined an embryonic TF circuitry that could be operative in TICs, resulting in up-regulation of oncogenes and loss of tumor suppressive functions. These OTBCs represent a patient-specific model system for the discovery of novel oncogenic targets in claudin-low tumors.
authors.affiliation: Department of Pharmacology, The University of North Carolina at Chapel Hill, 120 Mason Farm Road, Chapel Hill, NC 27599, USA.
authors.forename: Adriana S
authors.initials: AS
authors.lastname: Beltran
authors.forename: Ashley G
authors.initials: AG
authors.lastname: Rivenbark
authors.forename: Bryan T
authors.initials: BT
authors.lastname: Richardson
authors.forename: Xinni
authors.initials: X
authors.lastname: Yuan
authors.forename: Haili
authors.initials: H
authors.lastname: Quian
authors.forename: John P
authors.initials: JP
authors.lastname: Hunt
authors.forename: Eric
authors.initials: E
authors.lastname: Zimmerman
authors.forename: Lee M
authors.initials: LM
authors.lastname: Graves
authors.forename: Pilar
authors.initials: P
authors.lastname: Blancafort
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology; D002277:Carcinoma / Q000235:genetics / Q000473:pathology; D002454:Cell Differentiation / Q000235:genetics; D019070:Cell Lineage; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000473:pathology*; D016922:Cellular Senescence / Q000235:genetics; D004847:Epithelial Cells; D005260:Female; D005786:Gene Expression Regulation; D016147:Genes, Tumor Suppressor; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D042361:Mammary Glands, Human / Q000166:cytology / Q000378:metabolism / Q000473:pathology*; D008648:Mesoderm / Q000502:physiology; D051379:Mice; D008819:Mice, Nude; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics* / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D034741:RNA, Small Interfering; D055748:SOXB1 Transcription Factors / Q000235:genetics; D013234:Stem Cells / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D034741:RNA, Small Interfering; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D014157:Transcription Factors; C492123:ZIC1 protein, human