Acquisition of chemoresistance and EMT phenotype is linked with activation of the endothelin A receptor pathway in ovarian carcinoma cells.

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. The effects of ET-1 axis on cell proliferation, drug-induced apoptosis, invasiveness, 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. 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. 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.

Rosanò L ，Cianfrocca R ，Spinella F ，Di Castro V ，Nicotra MR ，Lucidi A ，Ferrandina G ，Natali PG ，Bagnato A ... -  《-》

Epithelial-mesenchymal transition in ovarian cancer progression: a crucial role for the endothelin axis.

In ovarian carcinoma, acquisition of invasiveness is accompanied by the loss of the epithelial features and the gain of a mesenchymal phenotype, a process known as epithelial-mesenchymal transition (EMT). The endothelin A receptor (ET(A)R)/endothelin-1 (ET-1) axis is overexpressed in primary and metastatic ovarian carcinoma. In this tumor type, the ET-1/ET(A)R axis has a critical role in ovarian carcinoma progression by inducing proliferation, survival, neoangiogenesis, loss of intercellular communication and invasion. Recently, we demonstrated that the ET-1/ET(A)R autocrine pathway drives EMT in ovarian tumor cells by inducing an invasive phenotype through downregulation of E-cadherin, increased levels of beta-catenin, Snail and other mesenchymal markers, and suppression of E-cadherin promoter activity. Activation of ET(A)R by ET-1 triggers a phosphatidylinositol 3-kinase-dependent integrin-linked kinase (ILK)-mediated signaling pathway leading to glycogen synthase kinase-3beta (GSK-3beta) inhibition, Snail and beta-catenin stabilization and transcriptional programs that control EMT. Transfection of dominant negative ILK or exposure to an ILK inhibitor suppresses the ET-1-induced phosphorylation of GSK-3beta as well as Snail and beta-catenin protein stability, transcriptional activity and invasiveness, indicating that ET-1/ET(A)R-induced EMT depends on ILK activity. ET(A)R blockade by specific antagonists, or reduction by ET(A)R RNA interference, reverses EMT and cell invasion by inhibiting autocrine signaling pathways. In ovarian carcinoma xenografts, the specific ET(A)R antagonist ABT-627 suppresses EMT determinants and tumor growth. In human ovarian cancers, ET(A)R expression is associated with E-cadherin downregulation, N-cadherin expression and tumor grade. In conclusion, our findings demonstrate that ET(A)R activation by ET-1 is a key mechanism of the complex signaling network that promotes EMT as well as ovarian cancer cell invasion. The small molecule ET(A)R antagonist achieves concomitant suppression of tumor growth and EMT effectors, providing a new opportunity for therapeutic intervention in which targeting ILK pathway and the related Snail and beta-catenin signaling cascade via ET(A)R blockade may be advantageous in the treatment of ovarian cancer.

Bagnato A ，Rosanò L 《-》

Inhibition of cyclooxygenase-1 and -2 expression by targeting the endothelin a receptor in human ovarian carcinoma cells.

New therapies against cancer are based on targeting cyclooxygenase (COX)-2. Activation of the endothelin A receptor (ET(A)R) by endothelin (ET)-1 is biologically relevant in several malignancies, including ovarian carcinoma. In this tumor, the ET-1/ET(A)R autocrine pathway promotes mitogenesis, apoptosis protection, invasion, and neoangiogenesis. Because COX-1 and COX-2 are involved in ovarian carcinoma progression, we investigated whether ET-1 induced COX-1 and COX-2 expression through the ET(A)R at the mRNA and protein level in HEY and OVCA 433 ovarian carcinoma cell lines by Northern blot, reverse transcription-PCR, Western blot, and immunohistochemistry; we also investigated the activity of the COX-2 promoter by luciferase assay and the release of prostaglandin (PG) E(2) by ELISA. ET-1 significantly increases the expression of COX-1 and COX-2, COX-2 promoter activity, and PGE(2) production. These effects depend on ET(A)R activation and involve multiple mitogen-activated protein kinase (MAPK) signaling pathways, including p42/44 MAPK, p38 MAPK, and transactivation of the epidermal growth factor receptor. COX-2 inhibitors and, in part, COX-1 inhibitor blocked ET-1-induced PGE(2) and vascular endothelial growth factor release, indicating that both enzymes participate in PGE(2) production to a different extent. Moreover, inhibition of human ovarian tumor growth in nude mice after treatment with the potent ET(A)R-selective antagonist ABT-627 is associated with reduced COX-2 and vascular endothelial growth factor expression. These results indicate that impairing COX-1 and COX-2 and their downstream effect by targeting ET(A)R can be therapeutically advantageous in ovarian carcinoma treatment. Pharmacological blockade of the ET(A)R is an attractive strategy to control COX-2 induction, which has been associated with ovarian carcinoma progression and chemoresistance.

Spinella F ，Rosanò L ，Di Castro V ，Nicotra MR ，Natali PG ，Bagnato A ... -  《CLINICAL CANCER RESEARCH》

Combination therapy of zibotentan with cisplatinum and paclitaxel is an effective regimen for epithelial ovarian cancer.

Rosanò L ，Cianfrocca R ，Spinella F ，Di Castro V ，Natali PG ，Bagnato A ... -  《-》

Endothelin-1 promotes epithelial-to-mesenchymal transition in human ovarian cancer cells.

Rosanò L ，Spinella F ，Di Castro V ，Nicotra MR ，Dedhar S ，de Herreros AG ，Natali PG ，Bagnato A ... -  《CANCER RESEARCH》