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Small Interfering RNA Molecules Targeting Endothelin-Converting Enzyme-1 Inhibit Endothelin-1 Synthesis and the Invasive Phenotype of Ovarian Carcinoma Cells

¹ Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel; ² Institut National de la Sante et de la Recherche Medicale U833, College de France, Paris, France; ³ Pathology Clinic, Norwegian Radium Hospital, Rikshospitalet Medical Center; ⁴ Faculty Division Radiumhospitalet, University of Oslo, Oslo, Norway; and ⁵ Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel

Requests for reprints: Rina Meidan, Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Animal Science Building, P. O. Box 12, Rehovot 76100, Israel. Phone: 972-89489394; Fax: 972-89465763; E-mail: rina.meidan{at}huji.ac.il.

Key Words: Endothelin-1 • ETA • Invasiveness

Endothelin-1 (ET-1) has been implicated in the progression of various cancers, including ovarian carcinoma. We found that the ovarian carcinoma cell lines ES2 and OVCAR3 and tumors from different anatomic sites expressed ET-1 system members [ET receptor A and ET-converting enzyme-1 (ECE-1)]. However, only ECE-1 was significantly higher in the solid tumors compared with effusions. We therefore investigated the effect of RNA interference-induced knockdown of ECE-1, the key enzyme in ET-1 production, on these two ovarian carcinoma cell lines. Small interfering RNA (siRNA) targeting of ECE-1 markedly reduced ECE-1 mRNA and protein levels, which subsequently led to 80% to 90% inhibition of ET-1 peptide secretion by the cells. ECE-1 silencing also profoundly affected the behavior of tumor cells compared with cells treated with scrambled siRNA. Silenced cells exhibited (a) reduced ET-1–dependent p44/42 mitogen-activated protein kinase phosphorylation; (b) decreased invasiveness and matrix metalloproteinase-2 activity; (c) improved adhesion to basal lamina proteins, laminin-1, and collagen IV; and (d) increased E-cadherin, an epithelial adhesion molecule, and reduced N-cadherin expression, a mesenchymal marker. Altered cell adherence is one of the hallmarks of the transformed phenotype, often characterized by the loss of the epithelial features and the gain of a mesenchymal phenotype. ECE-1 ablation did not, however, alter viable ovarian carcinoma cell numbers. Addition of exogenous ET-1 reversed the effects cited above. Taken together, these data indicate that siRNA is an effective tool for manipulating ECE-1 expression, ET-1 biosynthesis, and invasiveness of ovarian carcinoma. ECE-1 silencing may therefore develop into a promising novel anticancer therapy. [Cancer Res 2008;68(22):9265–73]