Cancer Therapy: Preclinical Acquisition of Chemoresistance and EMT Phenotype Is Linked with Activation of the Endothelin A Receptor Pathway in Ovarian Carcinoma Cells Laura Rosanò 1 , Roberta Cianfrocca 1 , Francesca Spinella 1 , Valeriana Di Castro 1 , Maria Rita Nicotra 2 , Alessandro Lucidi 3 , Gabriella Ferrandina 3 , Pier Giorgio Natali 1 , and Anna Bagnato 1 Abstract Purpose: Emerging evidence suggests molecular and phenotypic association between chemoresistance and epithelial–mesenchymal transition (EMT) in cancer. Endothelin-1 (ET-1)/endothelin A receptor (ET A R) axis is implicated in the pathobiology of epithelial ovarian cancer (EOC) by driving tumor- promoting effects, including EMT. Here, we analyzed how ET A R regulates chemoresistance and EMT in EOC. Experimental Design: The effects of ET-1 axis on cell proliferation, drug-induced apoptosis, invasive- ness, and EMT were analyzed in cultured EOC cells sensitive and resistant to cisplatinum and taxol. Tumor growth in response to ET A R antagonist was examined in EOC xenografts. ET A R expression was examined in 60 human EOC tumors by immunohistochemistry and correlated with chemoresistance and EMT. Results: In resistant EOC cells ET-1 and ET A R are upregulated, paralleled by enhanced mitogen activated protein kinase (MAPK) and Akt phosphorylation and cell proliferation. Moreover, in these cells the expression of E-cadherin transcriptional repressors, including Snail, Slug, and Twist, as well as of mesenchymal markers, such as vimentin and N-cadherin, were upregulated and linked with enhanced invasive behavior. Interestingly, ET A R blockade with zibotentan, a specific ET A R antagonist, or its silencing, downregulated Snail activity, restored drug sensitivity to cytotoxic-induced apoptosis, and inhibited the invasiveness of resistant cells. In vivo, zibotentan inhibited tumor growth of sensitive and resistant EOC xenografts, and sensitized to chemotherapy. Analysis of EOC human tissues revealed that ET A R is overexpressed in resistant tumors and is associated with EMT phenotype. Conclusions: Our data provide the first evidence that blockade of ET A R-driven EMT can overcome chemoresistance and inhibit tumor progression, improving the outcome of EOC patients’ treatment. Clin Cancer Res; 17(8); 2350–60. Ó2011 AACR. Introduction Ovarian cancer accounts for the highest tumor-related mortality in women with gynaecologic malignancy (1). The identification of the molecular mechanisms underlying chemoresistance is mandatory to achieve advancement in ovarian cancer therapy (2). Accumulating evidences demonstrated that epithelial–mesenchymal transition (EMT), which modulates cancer progression and metasta- sis, has also been implicated in the onset of drug resistance and tumor relapses, representing an escape mechanism from apoptosis (3). Therefore, the acquisition of mesench- ymal phenotypes engenders tumor cells with a multifaceted capacity to proliferate, migrate, and avoid cell death and permanent arrest, as well as protection from extracellular signals and drug effect activities (4). The hallmark of EMT is loss of the epithelial molecule E-cadherin and gain of mesenchymal markers, such as N-cadherin and vimentin. The E-cadherin repressors, Snail and Slug, which interact with E-box elements located within the proximal region of the E-cadherin promoter, and the basic helix-loop-helix transcription factor Twist, are significant inducers of EMT in cancer cells by repressing E-cadherin expression (5). Several clinical studies have shown that increased expres- sion of E-cadherin is associated with improved survival in several tumor types (6, 7), and silencing of E-cadherin transcriptional suppressors can increase cellular sensitivity to genotoxic stress (8). Since EMT development is driven by key modulators that are directly controlled by numerous extracellular signals and pathways (5), it is becoming clear that the blockade of these signaling pathways is critical for Authors' Affiliations: 1 Molecular Pathology Laboratory, Regina Elena National Cancer Institute; 2 Molecular Biology and Pathology Institute, National Research Council, Rome; and 3 Gynecologic Oncology Unit, Catholic University, Campobasso, Italy Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Anna Bagnato, Molecular Pathology Laboratory, Regina Elena National Cancer Institute, Via delle Messi d’Oro 156, 00158 Rome, Italy. Phone: 39-06-52662565; Fax: 39-06-52662600; E-mail: bagnato@ifo.it doi: 10.1158/1078-0432.CCR-10-2325 Ó2011 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 17(8) April 15, 2011 2350 Downloaded from http://aacrjournals.org/clincancerres/article-pdf/17/8/2350/2003514/2350.pdf by guest on 05 December 2023