Valeriana Di Castro

Endothelin-1 Induces an Angiogenic Phenotype in Cultured Endothelial Cells and Stimulates Neovascularization In Vivo

The endothelial cell-derived endothelin-1 (ET-1) is a potent mitogen for endothelial cells, vascular smooth muscle cells, and tumor cells. In this study, we analyzed the role of ET-1 on human umbilical vein endothelial cell (HUVEC) phenotype related to different stages of angiogenesis. ET-1 promoted HUVEC proliferation, migration, and invasion in a dose-dependent manner. The ET(B) receptor (ET(B)R) antagonist, BQ 788, blocked the angiogenic effects induced by ET-1, whereas the ET(A)R antagonist was less effective. ET-1 stimulated matrix metalloproteinase-2 mRNA expression and metalloproteinase-2 production, as determined by reverse transcriptase-polymerase chain reaction and gelatin zymography. Furthermore ET-1 was able to enhance HUVEC differentiation into cord vascular-like structures on Matrigel. When tested in combination with vascular endothelial growth factor (VEGF), ET-1 enhanced VEGF-induced angiogenic-related effects on endothelial cells in vitro. Finally, using the Matrigel plug neovascularization assay in vivo, ET-1 in combination with VEGF stimulated an angiogenic response comparable to that elicited by basic fibroblast growth factor. These findings demonstrated that ET-1 induces angiogenic responses in cultured endothelial cells through ET(B)R and that stimulates neovascularization in vivo in concert with VEGF. ET-1 and its receptors acting as angiogenic regulators might represent new targets for anti-angiogenic therapy.

Endothelin-1 Induces Vascular Endothelial Growth Factor by Increasing Hypoxia-inducible Factor-1α in Ovarian Carcinoma Cells

Angiogenesis is an essential prerequisite for tumor growth, invasion, and metastasis. In ovarian carcinoma cells, endothelin-1 (ET-1) stimulates the secretion of vascular endothelial growth factor (VEGF), a major mediator of tumor angiogenesis. In OVCA 433 and HEY ovarian carcinoma cell lines, ET-1 treatment increases VEGF mRNA expression and induces VEGF protein levels in a time- and dose-dependent fashion, and do so to a greater extent under hypoxic conditions. ET-1 also increases hypoxia-inducible factor-1α (HIF-1α) accumulation and activates the HIF-1 transcription complex under both normoxic and hypoxic conditions, suggesting a role for HIF-1 in the induction of VEGF expression. These effects are inhibited by the selective ETA receptor (ETAR) antagonist, BQ123. The ET-1-induced increase in HIF-1α protein levels is due to the enhanced HIF-1α stabilization. These results implicate HIF-1α in the induction of VEGF expression in ET-1-stimulated ovarian carcinoma cells, and provide a mechanism whereby ET-1 acting selectively through ETAR can interact with the HIF-1α-dependent machinery of angiogenesis. Our results suggest that new therapeutic strategies using specific ETAR antagonists could provide an additional approach to the treatment of ovarian carcinoma by inhibiting neovascularization as well as tumor cell growth.

Role of Endothelin-1 in Neovascularization of Ovarian Carcinoma

Endothelin-1 (ET-1) is overexpressed in ovarian carcinomas and acts, via ET(A) receptors (ET(A)R), as an autocrine growth factor. In this study we investigate the role of ET-1 in the neovascularization of ovarian carcinoma. Archival specimens of primary (n = 40) and metastatic (n = 8) ovarian tumors were examined by immunohistochemistry for angiogenic factor and receptor expression and for microvessel density using antibodies against CD31, ET-1, vascular endothelial growth factor (VEGF), and their receptors. ET-1 expression correlated with neovascularization and with VEGF expression. The localization of functional ET(A)R and ET(A)R mRNA expression, as detected by autoradiography and in situ hybridization, was evident in tumors and in intratumoral vessels, whereas ET(B)R were expressed mainly in endothelial cells. High levels of ET-1 were detected in the majority of ascitic fluids of patients with ovarian carcinoma and significantly correlated with VEGF ascitic concentration. Furthermore ET-1, through ET(A)R, stimulated VEGF production in an ovarian carcinoma cell line, OVCA 433, by an extent comparable to hypoxia. Finally, conditioned media from OVCA 433 as well as ascitic fluids caused an increase in endothelial cell migration and the ET-1 receptor blockade significantly inhibited this angiogenic response. These findings indicate that ET-1 could modulate tumor angiogenesis, acting directly and in part through VEGF.

Endothelin-1 Promotes Epithelial-to-Mesenchymal Transition in Human Ovarian Cancer Cells

Despite considerable efforts to improve early detection and advances in chemotherapy, metastatic relapses remain a major challenge in the management of ovarian cancer. The endothelin A receptor (ET(A)R)/endothelin-1 (ET-1) axis has been shown to have a significant role in ovarian carcinoma by promoting tumorigenesis. Here we show that the ET-1/ET(A)R autocrine pathway drives epithelial-to-mesenchymal transition (EMT) in ovarian tumor cells by inducing a fibroblastoid and invasive phenotype, down-regulation 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 an integrin-linked kinase (ILK)-mediated signaling pathway leading to glycogen synthase kinase-3beta (GSK-3beta) inhibition, Snail and beta-catenin stabilization, and regulation of 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, activity, and invasiveness, indicating that ET-1/ET(A)R-induced EMT-promoting effects depend on ILK. 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, ABT-627, a specific ET(A)R antagonist, suppresses EMT determinants and tumor growth. In human ovarian cancers, ET(A)R expression is associated with E-cadherin down-regulation, N-cadherin expression, and tumor grade. Collectively, these findings provide evidence of a critical role for the ET-1/ET(A)R axis during distinct steps of ovarian carcinoma progression and identify novel targets of therapeutic intervention.

β-Arrestin links endothelin A receptor to β-catenin signaling to induce ovarian cancer cell invasion and metastasis

The activation of endothelin-A receptor (ETAR) by endothelin-1 (ET-1) has a critical role in ovarian tumorigenesis and progression. To define the molecular mechanism in ET-1-induced tumor invasion and metastasis, we focused on β-arrestins as scaffold and signaling proteins of G protein-coupled receptors. Here, we demonstrate that, in ovarian cancer cells, β-arrestin is recruited to ETAR to form two trimeric complexes: one through the interaction with Src leading to epithelial growth factor receptor (EGFR) transactivation and β-catenin Tyr phosphorylation, and the second through the physical association with axin, contributing to release and inactivation of glycogen synthase kinase (GSK)-3β and β-catenin stabilization. The engagement of β-arrestin in these two signaling complexes concurs to activate β-catenin signaling pathways. We then demonstrate that silencing of both β-arrestin-1 and β-arrestin-2 inhibits ETAR-driven signaling, causing suppression of Src, mitogen-activated protein kinase (MAPK), AKT activation, as well as EGFR transactivation and a complete inhibition of ET-1-induced β-catenin/TCF transcriptional activity and cell invasion. ETAR blockade with the specific ETAR antagonist ZD4054 abrogates the engagement of β-arrestin in the interplay between ETAR and the β-catenin pathway in the invasive program. Finally, ETAR is expressed in 85% of human ovarian cancers and is preferentially co-expressed with β-arrestin-1 in the advanced tumors. In a xenograft model of ovarian metastasis, HEY cancer cells expressing β-arrestin-1 mutant metastasize at a reduced rate, highlighting the importance of this molecule in promoting metastases. ZD4054 treatment significantly inhibits metastases, suggesting that specific ETAR antagonists, by disabling multiple signaling activated by ETAR/β-arrestin, may represent new therapeutic opportunities for ovarian cancer.

Endothelin B Receptor Blockade Inhibits Dynamics of Cell Interactions and Communications in Melanoma Cell Progression

Phenotypic and genotypic analyses of cutaneous melanoma have identified the endothelin B receptor (ETBR) as tumor progression marker, thus representing a potential therapeutic target. Here, we demonstrate that activation of ETBR by endothelin-1 (ET-1) and ET-3 leads to loss of expression of the cell adhesion molecule E-cadherin and associated catenin proteins and gain of N-cadherin expression. Exposure of melanoma cells to ET-1 leads to a 60% inhibition in intercellular communication by inducing phosphorylation of gap junctional protein connexin 43. Additionally, activation of the ETBR pathway increases αvβ3 and α2β1 integrin expression and matrix metalloproteinase (MMP)-2 and MMP-9, membrane type-1-MMP activation, and tissue inhibitor MMP-2 secretion. The ETBR pathway results into the downstream activation of focal adhesion kinase and extracellular signal-regulated kinase 1/2 signaling pathways, which lead to enhanced cell proliferation, adhesion, migration, and MMP-dependent invasion. The small molecule A-192621, an orally bioavailable nonpeptide ETBR antagonist, significantly inhibits melanoma growth in nude mice. These findings demonstrate that ET-1 and ET-3 through ETBR activation trigger signaling pathways involved in events associated with disruption of normal host-tumor interactions and progression of cutaneous melanoma. Pharmacological interruption of ETBR signaling may represent a novel therapeutic strategy in the treatment of this malignancy.

Integrin-linked kinase functions as a downstream mediator of endothelin-1 to promote invasive behavior in ovarian carcinoma

The endothelin-1 (ET-1) axis represents a novel target in several malignancies, including ovarian carcinoma. Upon being activated, the endothelin A receptor (ETAR) mediates multiple tumor-promoting activities, including mitogenesis, escape from apoptosis, angiogenesis, metastasis-related protease activation, epithelial-mesenchymal transition, and invasion. Integrin-linked kinase (ILK) is a multidomain focal adhesion protein that conveys intracellular signaling elicited by β1-integrin and growth factor receptors. In this study, we investigate whether the signaling triggered by ETAR leading to an aggressive phenotype is mediated by an ILK-dependent mechanism. In HEY and OVCA 433 ovarian carcinoma cell lines, activation of ETAR by ET-1 enhances the expression of α2β1 and α3β1 integrins. ILK activity increases as ovarian cancer cells adhere to type I collagen through β1 integrin signaling, and do so to a greater extent on ET-1 stimulation. ET-1 increases ILK mRNA and protein expression and activity in a time- and concentration-dependent manner. An ILK small-molecule inhibitor (KP-392) or transfection with a dominant-negative ILK mutant effectively blocks the phosphorylation of downstream signals, Akt and glycogen synthase kinase-3β. The blockade of ET-1/ETAR-induced ILK activity results in an inhibition of matrix metalloproteinase activation as well as of cell motility and invasiveness in a phosphoinositide 3 kinase–dependent manner. In ovarian carcinoma xenografts, ABT-627, a specific ETAR antagonist, suppresses ILK expression, Akt and glycogen synthase kinase-3β phosphorylation, and tumor growth. These data show that ILK functions as a downstream mediator of the ET-1/ETAR axis to potentiate aggressive cellular behavior. Thus, the ILK-related signaling cascade can be efficiently targeted by pharmacologic blockade of ETAR. [Mol Cancer Ther 2006;5(4):833–42]

Endothelin-1 decreases gap junctional intercellular communication by inducing phosphorylation of connexin 43 in human ovarian carcinoma cells.

Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. Loss of gap junctional intercellular communication (GJIC) is critical for tumor progression by allowing the cells to escape growth control. Exposure of HEY and OVCA 433 ovarian carcinoma cell lines to ET-1 led to a 50–75% inhibition in intercellular communication and to a decrease in the connexin 43 (Cx43)-based gap junction plaques. To investigate the phosphorylation state of Cx43, ovarian carcinoma cell lysates were immunoprecipitated and transient tyrosine phosphorylation of Cx43 was detected in ET-1-treated cells. BQ 123, a selective ETAR antagonist, blocked the ET-1-induced Cx43 phosphorylation and cellular uncoupling. Gap junction closure was prevented by tyrphostin 25 and by the selective c-Src inhibitor, PP2. Furthermore, the increased Cx43 tyrosine phosphorylation was correlated with ET-1-induced increase of c-Src activity, and PP2 suppressed the ET-1-induced Cx43 tyrosine phosphorylation, indicating that inhibition of Cx43-based GJIC is mainly mediated by the Src tyrosine kinase pathway. In vivo, the inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation. These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression also by preventing the loss of GJIC.

Endothelin-1 and Endothelin-3 Promote Invasive Behavior via Hypoxia-Inducible Factor-1α in Human Melanoma Cells

Endothelin (ET) B receptor (ETBR), which is overexpressed in human cutaneous melanomas, promotes tumorigenesis upon activation by ET-1 or ET-3, thus representing a potential novel therapeutic target. Hypoxia-inducible factor-1α (HIF-1α) is the transcriptional factor that conveys signaling elicited by hypoxia and growth factor receptors. Here, we investigated the interplay between ET axis and hypoxia in primary and metastatic melanoma cell lines. We report that under normoxic conditions, ETBR activation by ET-1/ET-3 enhances vascular endothelial growth factor (VEGF) up-regulation, cyclooxygenase (COX)-1/COX-2 protein expression and COX-2 promoter activity, prostaglandin E2 (PGE2) production, and do so to a greater extent under hypoxia. Moreover, COX-1/COX-2 inhibitors block ET-induced PGE2 and VEGF secretion, matrix metalloproteinase (MMP) activation, and cell invasion, indicating that both enzymes function as downstream mediators of ET-induced invasive properties. The ETBR selective antagonist BQ788 or transfection with ETBR small interfering RNA (siRNA) block the ET-mediated effects. ETs also increase HIF-1α expression under both normoxic and hypoxic conditions and its silencing by siRNA desensitizes COX-2 transcriptional activity, PGE2 and VEGF production, and MMP activation in response to ET-3, implicating, for the first time, HIF-1α/COX as downstream targets of ETBR signaling leading to invasiveness. In melanoma xenografts, specific ETBR antagonist suppresses tumor growth, neovascularization, and invasiveness-related factors. Collectively, these results identify a new mechanism whereby ET-1/ET-3/ETBR axis can promote and interact with the HIF-1α–dependent machinery to amplify the COX-mediated invasive behavior of melanoma. New therapeutic strategies using specific ETBR antagonist could provide an improved approach to the treatment of melanoma by inhibiting tumor growth and progression. [Cancer Res 2007;67(4):1725–34]

Endothelin-1 Stimulates Lymphatic Endothelial Cells and Lymphatic Vessels to Grow and Invade

The lymphatic vasculature is essential for tissue fluid homeostasis and cancer metastasis, although the molecular mechanisms involved remain poorly characterized. Endothelin-1 (ET-1) axis plays a crucial role in angiogenesis and tumorigenesis. Here, we first report that ET-1 acts as a lymphangiogenic mediator. We performed in vitro and in vivo studies and show that lymphatic endothelial cells produce ET-1, ET-3, and express the endothelin B receptor (ET(B)R). In these cells, ET-1 promotes proliferation, invasiveness, vascular-like structures formation, and phosphorylation of AKT and p42/44 mitogen-activated protein kinase through ET(B)R. In normoxic conditions, ET-1 is also able to up-regulate the expression of vascular endothelial growth factor (VEGF)-C, VEGF receptor-3, and VEGF-A, and to stimulate hypoxia-inducible factor (HIF)-1alpha expression similarly to hypoxia. Moreover, HIF-1alpha silencing by siRNA desensitizes VEGF-C and VEGF-A production in response to ET-1 or hypoxia, implicating HIF-1alpha/VEGF as downstream signaling molecules of ET-1 axis. Double immunofluorescence analysis of human lymph nodes reveals that lymphatic vessels express ET(B)R together with the lymphatic marker podoplanin. Furthermore, a Matrigel plug assay shows that ET-1 promotes the outgrowth of lymphatic vessels in vivo. ET(B)R blockade with the specific antagonist, BQ788, inhibits in vitro and in vivo ET-1-induced effects, demonstrating that ET-1 through ET(B)R directly regulates lymphatic vessel formation and by interacting with the HIF-1alpha-dependent machinery can amplify the VEGF-mediated lymphatic vascularization. Our results suggest that ET-1 axis is indeed a new player in lymphangiogenesis and that targeting pharmacologically ET(B)R and related signaling cascade may be therapeutically exploited in a variety of diseases including cancer.