Endothelin-1/endothelin A receptor axis activates RhoA GTPase in epithelial ovarian cancer

Piera Tocci, Valentina Caprara, Roberta Cianfrocca, Rosanna Sestito, Valeriana Di Castro, Anna Bagnato, Laura Rosanò

Research output: Contribution to journalArticle

Abstract

AIMS: 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.

MAIN 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.

KEY FINDINGS: 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.

SIGNIFICANCE: Altogether these findings reveal a new pathway that depends on β-arr1 to sustain RhoA/ROCK signaling in response to ETAR activation in EOC.

Original languageEnglish
Pages (from-to)49-54
Number of pages6
JournalLife Sciences
Volume159
DOIs
Publication statusPublished - Aug 15 2016

Keywords

  • Cell Line, Tumor
  • Female
  • Humans
  • Neoplasms, Glandular and Epithelial
  • Ovarian Neoplasms
  • Receptor, Endothelin A
  • rhoA GTP-Binding Protein
  • Journal Article

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  • Cite this

    Tocci, P., Caprara, V., Cianfrocca, R., Sestito, R., Di Castro, V., Bagnato, A., & Rosanò, L. (2016). Endothelin-1/endothelin A receptor axis activates RhoA GTPase in epithelial ovarian cancer. Life Sciences, 159, 49-54. https://doi.org/10.1016/j.lfs.2016.01.008