title: Endothelin-1/endothelin A receptor axis activates RhoA GTPase in epithelial ovarian cancer.
abstract: OBJECTIVE
  The endothelin-1 (ET-1)/ET A receptor (ETAR) signaling pathway is critical driver of epithelial ovarian cancer (EOC) progression. Emerging evidences demonstrate that the scaffolding protein β-arrestin-1 (β-arr1) downstream of ETAR guides cell motility, although the signaling pathways by which ETAR activation controls these process are not well understood. Here, we set out to molecularly dissect whether RhoA GTPase activation is a mediator of ET-1 signaling controlling EOC cell migration.
  
  
  METHODS
  We cultured EOC cell lines (HEY, SKOV3, OVCAR, A2780 and 2008) with ET-1 and the ET-1R antagonist macitentan. RhoA expression was evaluated by RT-PCR. Activation of RhoA and ROCK1 was evaluated by pull down and kinase assays, respectively. Cell motility was evaluated by chemotaxis and wound healing assays, in untrasfected cells by using ROCK chemical inhibitors, Y-27632 or Fasudil, or in cells after transfection with dominant negative RhoA construct. The phosphorylation of myosin light chain 2 (MLC2) was evaluated by immunoblotting. Pseudopodia formation was evaluated by a pseudopodia kit assay.
  
  
  RESULTS
  In EOC cells, ET-1 activates RhoA and downstream ROCK1 and MLC2. These effects were inhibited by β-arr1 silencing, suggesting that ET-1/ETAR regulate RhoA signaling through β-arr1. At functional level, the activation of RhoA/ROCK signaling led to enhanced cell migration and pseudopodia formation. The suppressive effect of the ROCK inhibitors, as well as of macitentan, demonstrates that RhoA is involved in ET-1/ETAR-induced cell migration.
  
  
  CONCLUSIONS
  Altogether these findings reveal a new pathway that depends on β-arr1 to sustain RhoA/ROCK signaling in response to ETAR activation in EOC.
authors.affiliation: Translation Research Functional Area, Regina Elena National Cancer Institute Rome, Italy.
authors.forename: Piera
authors.initials: P
authors.lastname: Tocci
authors.affiliation: Translation Research Functional Area, Regina Elena National Cancer Institute Rome, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Caprara
authors.affiliation: Translation Research Functional Area, Regina Elena National Cancer Institute Rome, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Cianfrocca
authors.affiliation: Translation Research Functional Area, Regina Elena National Cancer Institute Rome, Italy.
authors.forename: Rosanna
authors.initials: R
authors.lastname: Sestito
authors.affiliation: Translation Research Functional Area, Regina Elena National Cancer Institute Rome, Italy.
authors.forename: Valeriana
authors.initials: V
authors.lastname: Di Castro
authors.affiliation: Translation Research Functional Area, Regina Elena National Cancer Institute Rome, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Bagnato
authors.affiliation: Translation Research Functional Area, Regina Elena National Cancer Institute Rome, Italy. Electronic address: rosano@ifo.it.
authors.forename: Laura
authors.initials: L
authors.lastname: Rosanò
mesh_terms: D000077216:Carcinoma, Ovarian Epithelial; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D009375:Neoplasms, Glandular and Epithelial / Q000201:enzymology*; D010051:Ovarian Neoplasms / Q000201:enzymology*; D044022:Receptor, Endothelin A / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D044022:Receptor, Endothelin A; D020742:rhoA GTP-Binding Protein
keywords: Endothelin; Ovarian cancer; RhoA