title: Exosome-mediated microRNA signaling from breast cancer cells is altered by the anti-angiogenesis agent docosahexaenoic acid (DHA).
abstract: BACKGROUND
  Docosahexaenoic acid (DHA) is a natural compound with anticancer and anti-angiogenesis activity that is currently under investigation as both a preventative agent and an adjuvant to breast cancer therapy. However, the precise mechanisms of DHA's anticancer activities are unclear. It is understood that the intercommunication between cancer cells and their microenvironment is essential to tumor angiogenesis. Exosomes are extracellular vesicles that are important mediators of intercellular communication and play a role in promoting angiogenesis. However, very little is known about the contribution of breast cancer exosomes to tumor angiogenesis or whether exosomes can mediate DHA's anticancer action.
  
  
  RESULTS
  Exosomes were collected from MCF7 and MDA-MB-231 breast cancer cells after treatment with DHA. We observed an increase in exosome secretion and exosome microRNA contents from the DHA-treated cells. The expression of 83 microRNAs in the MCF7 exosomes was altered by DHA (>2-fold). The most abundant exosome microRNAs (let-7a, miR-23b, miR-27a/b, miR-21, let-7, and miR-320b) are known to have anti-cancer and/or anti-angiogenic activity. These microRNAs were also increased by DHA treatment in the exosomes from other breast cancer lines (MDA-MB-231, ZR751 and BT20), but not in exosomes from normal breast cells (MCF10A). When DHA-treated MCF7 cells were co-cultured with or their exosomes were directly applied to endothelial cell cultures, we observed an increase in the expression of these microRNAs in the endothelial cells. Furthermore, overexpression of miR-23b and miR-320b in endothelial cells decreased the expression of their pro-angiogenic target genes (PLAU, AMOTL1, NRP1 and ETS2) and significantly inhibited tube formation by endothelial cells, suggesting that the microRNAs transferred by exosomes mediate DHA's anti-angiogenic action. These effects could be reversed by knockdown of the Rab GTPase, Rab27A, which controls exosome release.
  
  
  CONCLUSIONS
  We conclude that DHA alters breast cancer exosome secretion and microRNA contents, which leads to the inhibition of angiogenesis. Our data demonstrate that breast cancer exosome signaling can be targeted to inhibit tumor angiogenesis and provide new insight into DHA's anticancer action, further supporting its use in cancer therapy.
authors.affiliation: Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA. bethany-hannafon@ouhsc.edu.
authors.forename: Bethany N
authors.initials: BN
authors.lastname: Hannafon
authors.affiliation: Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA. 5karlajo5@gmail.com.
authors.forename: Karla J
authors.initials: KJ
authors.lastname: Carpenter
authors.affiliation: Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA. william-berry@ouhsc.edu.
authors.forename: William L
authors.initials: WL
authors.lastname: Berry
authors.affiliation: Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA. ralf-janknecht@ouhsc.edu.|Peggy and Charles Stephenson Cancer Center, Oklahoma City, OK, 73104, USA. ralf-janknecht@ouhsc.edu.
authors.forename: Ralf
authors.initials: R
authors.lastname: Janknecht
authors.affiliation: Department of Surgery, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA. william-dooley@ouhsc.edu.|Peggy and Charles Stephenson Cancer Center, Oklahoma City, OK, 73104, USA. william-dooley@ouhsc.edu.
authors.forename: William C
authors.initials: WC
authors.lastname: Dooley
authors.affiliation: Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA. weiqun-ding@ouhsc.edu.|Peggy and Charles Stephenson Cancer Center, Oklahoma City, OK, 73104, USA. weiqun-ding@ouhsc.edu.
authors.forename: Wei-Qun
authors.initials: WQ
authors.lastname: Ding
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D001692:Biological Transport; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D004281:Docosahexaenoic Acids / Q000494:pharmacology*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D055354:Exosomes / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000235:genetics; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000187:drug effects*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000075745:rab27 GTP-Binding Proteins
chemical_list: D020533:Angiogenesis Inhibitors; D035683:MicroRNAs; D042461:Vascular Endothelial Growth Factor A; D000075745:rab27 GTP-Binding Proteins; D004281:Docosahexaenoic Acids; C066694:RAB27A protein, human; D020691:rab GTP-Binding Proteins