title: miR-720 is a downstream target of an ADAM8-induced ERK signaling cascade that promotes the migratory and invasive phenotype of triple-negative breast cancer cells.
abstract: BACKGROUND
  ADAM8 (a disintegrin and metalloproteinase 8) protein promotes the invasive and metastatic phenotype of triple-negative breast cancer (TNBC) cells. High ADAM8 expression in breast cancer patients is an independent predictor of poor prognosis. Here, we investigated whether ADAM8 regulates specific miRNAs, their roles in aggressive phenotype, and potential use as biomarkers of disease.
  
  
  METHODS
  Microarray analysis was performed on RNA from MDA-MB-231 cells after transient ADAM8 knockdown using TaqMan miRNA cards. Changes in miRNA levels were confirmed using two ADAM8 siRNAs in TNBC cell lines. Kinase inhibitors, β1-integrin antagonist antibody, and different forms of ADAM8 were employed to elucidate the signaling pathway required for miR-720 expression. miR-720 levels were modulated using a specific antagomiR or a mimic, and effects on aggressive phenotype of TNBC cells were determined using Boyden chamber and 3D-Matrigel outgrowth assays. Plasma was isolated from mice before and after implantation of MDA-MB-231 cells and analyzed for miR-720 levels. Serum samples of TNBC patients were evaluated for their ADAM8 and miR-720 levels.
  
  
  RESULTS
  We identified 68 miRNAs differentially regulated upon ADAM8 knockdown, including decreased levels of secreted miR-720. Ectopic overexpression of wild-type ADAM8 or forms that lack metalloproteinase activity similarly induced miR-720 levels. The disintegrin and cysteine-rich domains of ADAM8 were shown to induce miR-720 via activation of a β1-integrin to ERK signaling cascade. Knockdown of miR-720 led to a significant decrease in migratory and invasive abilities of TNBC cells. Conversely, miR-720 overexpression rescued these properties. A profound increase in plasma levels of miR-720 was detected 7 days after TNBC cell inoculation into mouse mammary fat pads when tumors were barely palpable. Concordantly, miR-720 levels were found to be significantly higher in serum samples of TNBC patients with high ADAM8 expression.
  
  
  CONCLUSIONS
  We have shown for the first time that miR-720 is induced by ADAM8 signaling via ERK and plays an essential role in promoting the aggressive phenotype of TNBCs. miR-720 is elevated in serum of patients with ADAM8-high TNBC and, in a group with other miRNAs downstream of ADAM8, holds promise as a biomarker for early detection of or treatment response of ADAM8-positive TNBCs.
authors.affiliation: Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA, 02111, USA.
authors.forename: Sonia G
authors.initials: SG
authors.lastname: Das
authors.affiliation: Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA, 02111, USA.|Present address: Institut Curie, Centre de Recherche, UMR 144, 26 Rue d'Ulm, 75248, Paris, France.
authors.forename: Mathilde
authors.initials: M
authors.lastname: Romagnoli
authors.affiliation: Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA, 02111, USA.
authors.forename: Nora D
authors.initials: ND
authors.lastname: Mineva
authors.affiliation: INSERM U892, IRT-UN, 8 quai Moncousu, 44007, Nantes Cedex, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Barillé-Nion
authors.affiliation: INSERM U892, IRT-UN, 8 quai Moncousu, 44007, Nantes Cedex, France.|Institut de Cancérologie de Nantes, Centre de Lutte Contre le Cancer René Gauducheau, Boulevard Jacques Monod, 44 805, Saint-Herblain-Nantes Cedex, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Jézéquel
authors.affiliation: INSERM U892, IRT-UN, 8 quai Moncousu, 44007, Nantes Cedex, France.|Institut de Cancérologie de Nantes, Centre de Lutte Contre le Cancer René Gauducheau, Boulevard Jacques Monod, 44 805, Saint-Herblain-Nantes Cedex, France.
authors.forename: Mario
authors.initials: M
authors.lastname: Campone
authors.affiliation: Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA, 02111, USA. gail.sonenshein@tufts.edu.
authors.forename: Gail E
authors.initials: GE
authors.lastname: Sonenshein
mesh_terms: D051722:ADAM Proteins / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation / Q000235:genetics; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D020935:MAP Kinase Signaling System; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D035683:MicroRNAs / Q000096:biosynthesis* / Q000235:genetics; D009361:Neoplasm Invasiveness / Q000235:genetics*; D034741:RNA, Small Interfering; D064726:Triple Negative Breast Neoplasms / Q000235:genetics* / Q000473:pathology
chemical_list: C568636:MIRN720 microRNA, human; D008565:Membrane Proteins; D035683:MicroRNAs; D034741:RNA, Small Interfering; D051722:ADAM Proteins; C068490:ADAM8 protein, human
keywords: ADAM8; ERK; Triple-negative breast cancer; miR-720; miRNA