LMNA knock-down affects differentiation and progression of human neuroblastoma cells.

BACKGROUND: Neuroblastoma (NB) is one of the most aggressive tumors that occur in childhood. Although genes, such as MYCN, have been shown to be involved in the aggressiveness of the disease, the identification of new biological markers is still desirable. The induction of differentiation is one of the strategies used in the treatment of neuroblastoma. A-type lamins are components of the nuclear lamina and are involved in differentiation. We studied the role of Lamin A/C in the differentiation and progression of neuroblastoma. METHODOLOGY/PRINCIPAL FINDINGS: Knock-down of Lamin A/C (LMNA-KD) in neuroblastoma cells blocked retinoic acid-induced differentiation, preventing neurites outgrowth and the expression of neural markers. The genome-wide gene-expression profile and the proteomic analysis of LMNA-KD cells confirmed the inhibition of differentiation and demonstrated an increase of aggressiveness-related genes and molecules resulting in augmented migration/invasion, and increasing the drug resistance of the cells. The more aggressive phenotype acquired by LMNA-KD cells was also maintained in vivo after injection into nude mice. A preliminary immunohistochemistry analysis of Lamin A/C expression in nine primary stages human NB indicated that this protein is poorly expressed in most of these cases. CONCLUSIONS/SIGNIFICANCE: We demonstrated for the first time in neuroblastoma cells that Lamin A/C plays a central role in the differentiation, and that the loss of this protein gave rise to a more aggressive tumor phenotype.

Cell-to-cell signaling influences the fate of prostate cancer stem cells and their potential to generate more aggressive tumors.

An increasing number of malignancies has been shown to be initiated and propelled by small subpopulations of cancer stem cells (CSC). However, whether tumor aggressiveness is driven by CSC and by what extent this property may be relevant within the tumor mass is still unsettled. To address this issue, we isolated a rare tumor cell population on the basis of its CD44(+)CD24(-) phenotype from the human androgen-independent prostate carcinoma cell line DU145 and established its CSC properties. The behavior of selected CSC was investigated with respect to the bulk DU145 cells. The injection of CSC in nude mice generated highly vascularized tumors infiltrating the adjacent tissues, showing high density of neuroendocrine cells and expressing low levels of E-cadherin and ß-catenin as well as high levels of vimentin. On the contrary, when a comparable number of unsorted DU145 cells were injected the resulting tumors were less aggressive. To investigate the different features of tumors in vivo, the influence of differentiated tumor cells on CSC was examined in vitro by growing CSC in the absence or presence of conditioned medium from DU145 cells. CSC grown in permissive conditions differentiated into cell populations with features similar to those of cells held in aggressive tumors generated from CSC injection. Differently, conditioned medium induced CSC to differentiate into a cell phenotype comparable to cells of scarcely aggressive tumors originated from bulk DU145 cell injection. These findings show for the first time that CSC are able to generate differentiated cells expressing either highly or scarcely aggressive phenotype, thus influencing prostate cancer progression. The fate of CSC was determined by signals released from tumor environment. Moreover, using microarray analysis we selected some molecules which could be involved in this cell-to-cell signaling, hypothesizing their potential value for prognostic or therapeutic applications.

Human leukocyte antigen E contributes to protect tumor cells from lysis by natural killer cells.

The nonclassic class I human leukocyte antigen E (HLA-E) molecule engages the inhibitory NKG2A receptor on several cytotoxic effectors, including natural killer (NK) cells. Its tissue distribution was claimed to be wider in normal than in neoplastic tissues, and surface HLA-E was undetectable in most tumor cell lines. Herein, these issues were reinvestigated taking advantage of HLA-E-specific antibodies, immunohistochemistry, and biochemical methods detecting intracellular and surface HLA-E regardless of conformation. Contrary to published evidence, HLA-E was detected in a few normal epithelia and in a large fraction (approximately 1/3) of solid tumors, including those derived from HLA-E-negative/low-normal counterparts. Remarkably, HLA-E was detected in 30 of 30 tumor cell lines representative of major lymphoid and nonlymphoid lineages, and in 11 of 11, it was surface-expressed, although in a conformation poorly reactive with commonly used antibodies. Coexpression of HLA-E and HLA class I ligand donors was not required for surface expression but was associated with NKG2A-mediated protection from lysis by the cytotoxic cell line NKL and polyclonal NK cells from healthy donors, as demonstrated by antibody-mediated relief of protection in 10% to 20% of the tested target-effector combinations. NKG2A-mediated protection of additional targets became evident on NK effector blocking with antibodies to activating receptors (DNAM-1, natural cytotoxicity receptors, and NKG2D). Thus, initial evidence that the long-elusive HLA-E molecule is enhanced by malignant transformation and is functional in tumor cells is presented here, although its importance and precise functional role remain to be addressed in the context of a general understanding of the NK ligand-receptor network.

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

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, 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. 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.

Endothelin-1 inhibits prolyl hydroxylase domain 2 to activate hypoxia-inducible factor-1alpha in melanoma cells.

BACKGROUND: The endothelin B receptor (ET(B)R) promotes tumorigenesis and melanoma progression through activation by endothelin (ET)-1, thus representing a promising therapeutic target. The stability of hypoxia-inducible factor (HIF)-1alpha is essential for melanomagenesis and progression, and is controlled by site-specific hydroxylation carried out by HIF-prolyl hydroxylase domain (PHD) and subsequent proteosomal degradation. PRINCIPAL FINDINGS: Here we found that in melanoma cells ET-1, ET-2, and ET-3 through ET(B)R, enhance the expression and activity of HIF-1alpha and HIF-2alpha that in turn regulate the expression of vascular endothelial growth factor (VEGF) in response to ETs or hypoxia. Under normoxic conditions, ET-1 controls HIF-alpha stability by inhibiting its degradation, as determined by impaired degradation of a reporter gene containing the HIF-1alpha oxygen-dependent degradation domain encompassing the PHD-targeted prolines. In particular, ETs through ET(B)R markedly decrease PHD2 mRNA and protein levels and promoter activity. In addition, activation of phosphatidylinositol 3-kinase (PI3K)-dependent integrin linked kinase (ILK)-AKT-mammalian target of rapamycin (mTOR) pathway is required for ET(B)R-mediated PHD2 inhibition, HIF-1alpha, HIF-2alpha, and VEGF expression. At functional level, PHD2 knockdown does not further increase ETs-induced in vitro tube formation of endothelial cells and melanoma cell invasiveness, demonstrating that these processes are regulated in a PHD2-dependent manner. In human primary and metastatic melanoma tissues as well as in cell lines, that express high levels of HIF-1alpha, ET(B)R expression is associated with low PHD2 levels. In melanoma xenografts, ET(B)R blockade by ET(B)R antagonist results in a concomitant reduction of tumor growth, angiogenesis, HIF-1alpha, and HIF-2alpha expression, and an increase in PHD2 levels. CONCLUSIONS: In this study we identified the underlying mechanism by which ET-1, through the regulation of PHD2, controls HIF-1alpha stability and thereby regulates angiogenesis and melanoma cell invasion. These results further indicate that targeting ET(B)R may represent a potential therapeutic treatment of melanoma by impairing HIF-1alpha stability.

NF-kappaB, and not MYCN, regulates MHC class I and endoplasmic reticulum aminopeptidases in human neuroblastoma cells.

Neuroblastoma (NB) is the most common solid extracranial cancer of childhood. Amplification and overexpression of the MYCN oncogene characterize the most aggressive forms and are believed to severely downregulate MHC class I molecules by transcriptional inhibition of the p50 NF-kappaB subunit. In this study, we found that in human NB cell lines, high MYCN expression is not responsible for low MHC class I expression because neither transfection-mediated overexpression nor small interfering RNA suppression of MYCN affects MHC class I and p50 levels. Furthermore, we identified NF-kappaB as the immediate upstream regulator of MHC class I because the p65 NF-kappaB subunit binds MHC class I promoter in chromatin immunoprecipitation experiments, and MHC class I expression is enhanced by p65 transfection and reduced by (a) the chemical NF-kappaB inhibitor sulfasalazine, (b) a dominant-negative IKBalpha gene, and (c) p65 silencing. Moreover, we showed that the endoplasmic reticulum aminopeptidases ERAP1 and ERAP2, which generate MHC class I binding peptides, are regulated by NF-kappaB, contain functional NF-kappaB-binding elements in their promoters, and mimic MHC class I molecules in the expression pattern. Consistent with these findings, nuclear p65 was detected in NB cells that express MHC class I molecules in human NB specimens. Thus, the coordinated downregulation of MHC class I, ERAP1, and ERAP2 in aggressive NB cells is attributable to a low transcriptional availability of NF-kappaB, possibly due to an unknown suppressor other than MYCN.

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.

Beta-arrestin links endothelin A receptor to beta-catenin signaling to induce ovarian cancer cell invasion and metastasis.

The activation of endothelin-A receptor (ET(A)R) 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 beta-arrestins as scaffold and signaling proteins of G protein-coupled receptors. Here, we demonstrate that, in ovarian cancer cells, beta-arrestin is recruited to ET(A)R to form two trimeric complexes: one through the interaction with Src leading to epithelial growth factor receptor (EGFR) transactivation and beta-catenin Tyr phosphorylation, and the second through the physical association with axin, contributing to release and inactivation of glycogen synthase kinase (GSK)-3beta and beta-catenin stabilization. The engagement of beta-arrestin in these two signaling complexes concurs to activate beta-catenin signaling pathways. We then demonstrate that silencing of both beta-arrestin-1 and beta-arrestin-2 inhibits ET(A)R-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 beta-catenin/TCF transcriptional activity and cell invasion. ET(A)R blockade with the specific ET(A)R antagonist ZD4054 abrogates the engagement of beta-arrestin in the interplay between ET(A)R and the beta-catenin pathway in the invasive program. Finally, ET(A)R is expressed in 85% of human ovarian cancers and is preferentially co-expressed with beta-arrestin-1 in the advanced tumors. In a xenograft model of ovarian metastasis, HEY cancer cells expressing beta-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 ET(A)R antagonists, by disabling multiple signaling activated by ET(A)R/beta-arrestin, may represent new therapeutic opportunities for ovarian cancer.

Progression of colorectal cancers correlates with overexpression and loss of polarization of expression of the htid-1 tumor suppressor.

Recently, we identified htid-1, the human counterpart of the Drosophila tumor suppressor gene lethal(2)tumorous imaginal discs [l(2)tid], as a direct molecular ligand of the adenomatous polyposis coli (APC) tumor suppressor. The gene encodes three cytosolic (Tid50, Tid48 and Tid46) and three mitochondrial (Tid43, Tid40 and Tid38) proteins. In the colorectal epithelium the cytosolic forms hTid50/hTid48 interact under physiological conditions with the N-terminal region of APC. This complex which associates with additional proteins such as Hsp70, Hsc70, Actin, Dvl and Axin defines a novel physiological state of APC unrelated to beta-catenin degradation. Here we show that the expression of the genes htid-1 and APC was altered in colorectal tumors. These changes concerned both the localization and the expression level of all three htid-1 splice variants and of APC. Furthermore, we showed that the protein products of the two tumor suppressors co-localized in the basal and apical region of normal colon epithelia and that loss of differentiation capacity of colorectal cancers correlated with a shift in their expression patterns from compartmentalized to diffuse cytoplasmic. These findings support our hypothesis that the building of the multi-component complex mentioned above is associated with the maintenance of the polarity of cells and tissues. In addition, we provide evidence that colon cancer progression correlates with up-regulation of htid-1 and its ligand Hsp70. Since the Tid proteins are members of the DnaJ-like protein family, an essential component of the Hsp70/Hsc70 chaperone machinery, our findings describe a novel, causal link between the function of chaperone machines, APC-mediated Wg/Wnt signaling and tumor development.

ZD4054, a specific antagonist of the endothelin A receptor, inhibits tumor growth and enhances paclitaxel activity in human ovarian carcinoma in vitro and in vivo.

The autocrine endothelin (ET)-1/endothelin A receptor (ET(A)R) pathway is an important regulator of several processes involved in ovarian cancer progression, and its overexpression is associated with aggressive disease. These features have led to the proposal of the ET(A)R receptor as a potential target for improving ovarian cancer treatment. In this study, we evaluated in vitro and in vivo the effects of ZD4054, an orally active antagonist that specifically binds ET(A)R, as monotherapy, and in combination with paclitaxel. In the human ovarian cancer ET(A)R-positive cell lines HEY, OVCA 433, SKOV-3, and A-2780, ZD4054 effectively inhibited the basal and ET-1-induced cell proliferation, associated with the inhibition of AKT and p42/44MAPK phosphorylation, and with increased apoptosis, through the inhibition of bcl-2 and activation of caspase-3 and poly(ADP-ribose) polymerase proteins. ZD4054 treatment also resulted in a reduction of ET(A)R-driven angiogenesis and invasive mediators, such as vascular endothelial growth factor, cyclooxygenase-1/2, and matrix metalloproteinase (MMP). The combination of ZD4054 and paclitaxel led to the potentiation of all these effects, indicating that ZD4054, by blocking the ET(A)R-dependent proliferative, invasive, and antiapoptotic signals, can enhance sensitivity to paclitaxel. In HEY ovarian cancer xenografts, ZD4054 significantly inhibited tumor growth to the same degree as paclitaxel. Furthermore, ZD4054-dependent tumor growth inhibition was associated with a reduction in proliferation index, microvessel density, and MMP-2 expression. Interestingly, the combination of ZD4054 and paclitaxel produced additive antitumor effects, with 40% of mice remaining tumor-free, supporting a rationale for the clinical use of ZD4054 as monotherapy or in combination with cytotoxic drugs.

Combined targeting of endothelin A receptor and epidermal growth factor receptor in ovarian cancer shows enhanced antitumor activity.

Ovarian carcinomas overexpress endothelin A receptors (ET(A)R) and epidermal growth factor (EGF) receptor (EGFR). In these cells, endothelin-1 (ET-1) triggers mitogenic and invasive signaling pathways that are in part mediated by EGFR transactivation. Combined targeting of ET(A)R, by the specific ET(A)R antagonist ZD4054, and of EGFR by the EGFR inhibitor gefitinib (IRESSA), may offer improvements in ovarian carcinoma treatment. In HEY and OVCA 433 ovarian carcinoma cells, ET-1 or EGF induced rapid activation of EGFR, p42/44 mitogen-activated protein kinase (MAPK), and AKT. ZD4054 was able to reduce the ET-1-induced EGFR transactivation. Gefitinib significantly inhibited EGF- and ET-1-induced EGFR phosphorylation, but incompletely reduced the ET-1-induced activation of downstream targets. ZD4054 plus gefitinib resulted in a greater inhibition of EGFR, MAPK, and AKT phosphorylation, indicating the critical role of these interconnected signaling proteins. ZD4054 effectively inhibited cell proliferation, invasiveness, and vascular endothelial growth factor (VEGF) secretion. Concomitantly, ZD4054 enhanced apoptosis and E-cadherin promoter activity and expression. In both cell lines, the drug combination resulted in a significant decrease in cell proliferation (65%), invasion (52%), and VEGF production (50%), accompanied by a 2-fold increase in apoptosis. The coadministration of ZD4054 enhanced the efficacy of gefitinib leading to partial (82%) or complete tumor regression on HEY ovarian carcinoma xenografts. Antitumor effects were paralleled by biochemical and immunohistologic evidence of decreased vascularization, Ki-67, matrix metalloproteinase-2 (MMP-2), VEGF, MAPK and EGFR, and enhanced E-cadherin expression. The cross-signaling between the EGFR/ET(A)R pathways provides a rationale to combine EGFR inhibitors with ET(A)R antagonists, identifying new effective therapeutic opportunities for ovarian cancer.

Molecular cloning of hMena (ENAH) and its splice variant hMena+11a: epidermal growth factor increases their expression and stimulates hMena+11a phosphorylation in breast cancer cell lines.

hMena (ENAH), an actin regulatory protein involved in the control of cell motility and adhesion, is modulated during human breast carcinogenesis. In fact, whereas undetectable in normal mammary epithelium, hMena becomes overexpressed in high-risk benign lesions and primary and metastatic tumors. In vivo, hMena overexpression correlates with the HER-2(+)/ER(-)/Ki67(+) unfavorable prognostic phenotype. In vitro, neuregulin-1 up-regulates whereas Herceptin treatment down-modulates hMena expression, suggesting that it may couple tyrosine kinase receptor signaling to the actin cytoskeleton. Herein, we report the cloning of hMena and of a splice variant, hMena(+11a), which contains an additional exon corresponding to 21 amino acids located in the EVH2 domain, from a breast carcinoma cell line of epithelial phenotype. Whereas hMena overexpression consistently characterizes the transformed phenotype of tumor cells of different lineages, hMena(+11a) isoform is concomitantly present only in epithelial tumor cell lines. In breast cancer cell lines, epidermal growth factor (EGF) treatment promotes concomitant up-regulation of hMena and hMena(+11a), resulting in an increase of the fraction of phosphorylated hMena(+11a) isoform only. hMena(+11a) overexpression and phosphorylation leads to increased p42/44 mitogen-activated protein kinase (MAPK) activation and cell proliferation as evidenced in hMena(+11a)-transfected breast cancer cell lines. On the contrary, hMena knockdown induces reduction of p42/44 MAPK phosphorylation and of the proliferative response to EGF. The present data provide new insight into the relevance of actin cytoskeleton regulatory proteins and, in particular, of hMena isoforms in coupling multiple signaling pathways involved in breast cancer.

Endothelin-1 and endothelin-3 promote invasive behavior via hypoxia-inducible factor-1alpha in human melanoma cells.

Endothelin (ET) B receptor (ET(B)R), 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-1alpha (HIF-1alpha) 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, ET(B)R 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 E(2) (PGE(2)) production, and do so to a greater extent under hypoxia. Moreover, COX-1/COX-2 inhibitors block ET-induced PGE(2) and VEGF secretion, matrix metalloproteinase (MMP) activation, and cell invasion, indicating that both enzymes function as downstream mediators of ET-induced invasive properties. The ET(B)R selective antagonist BQ788 or transfection with ET(B)R small interfering RNA (siRNA) block the ET-mediated effects. ETs also increase HIF-1alpha expression under both normoxic and hypoxic conditions and its silencing by siRNA desensitizes COX-2 transcriptional activity, PGE(2) and VEGF production, and MMP activation in response to ET-3, implicating, for the first time, HIF-1alpha/COX as downstream targets of ET(B)R signaling leading to invasiveness. In melanoma xenografts, specific ET(B)R antagonist suppresses tumor growth, neovascularization, and invasiveness-related factors. Collectively, these results identify a new mechanism whereby ET-1/ET-3/ET(B)R axis can promote and interact with the HIF-1alpha-dependent machinery to amplify the COX-mediated invasive behavior of melanoma. New therapeutic strategies using specific ET(B)R antagonist could provide an improved approach to the treatment of melanoma by inhibiting tumor growth and progression.

Green tea polyphenol epigallocatechin-3-gallate inhibits the endothelin axis and downstream signaling pathways in ovarian carcinoma.

The polyphenol epigallocatechin-3-gallate (EGCG), the principal mediator of the green tea, has been known to possess antitumor effect. The endothelin A receptor (ET(A)R)/endothelin-1 (ET-1) axis is overexpressed in ovarian carcinoma representing a novel therapeutic target. In this study, we examined the green tea and EGCG effects on two ovarian carcinoma cell lines, HEY and OVCA 433. EGCG inhibited ovarian cancer cell growth and induced apoptosis that was associated with a decrease in Bcl-X(L) expression and activation of caspase-3. Treatment with green tea or EGCG inhibited ET(A)R and ET-1 expression and reduced the basal and ET-1-induced cell proliferation and invasion. The EGCG-induced inhibitory effects were associated with a decrease of ET(A)R-dependent activation of the p42/p44 and p38 mitogen-activated protein kinases and phosphatidylinositol 3-kinase pathway. Remarkably, EGCG treatment resulted in a lowering of basal and ET-1-induced angiogenesis and invasiveness mediators, such as vascular endothelial growth factor and tumor proteinase activation. Finally, in HEY ovarian carcinoma xenografts, tumor growth was significantly inhibited by oral administration of green tea. This effect was associated with a reduction in ET-1, ET(A)R, and vascular endothelial growth factor expression, microvessel density, and proliferation index. These results provide a novel insight into the mechanism by which EGCG, affecting multiple ET(A)R-dependent pathways, may inhibit ovarian carcinoma growth, suggesting that EGCG may be useful in preventing and treating ovarian carcinoma in which ET(A)R activation by ET-1 plays a critical role in tumor growth and progression.

Endothelin-1 is required during epithelial to mesenchymal transition in ovarian cancer progression.

In a range of human cancers, tumorigenesis is promoted by activation of the endothelin A receptor (ET(A)R)/endothelin-1 (ET-1) axis. ET-1 and ET(A)R are overexpressed in primary and metastatic ovarian carcinomas, and high levels of ET-1 are detectable in patient ascites, suggesting that ET-1 may promote tumor dissemination. Moreover, in these tumors, engagement of ET(A) receptor by ET-1 triggers tumor growth, survival, angiogenesis, and invasiveness. Thus, ET-1 enhances the secretion of matrix metalloproteinases, disrupts intercellular communications, and stimulates cell migration and invasion. Therefore, we investigated the role of the ET-1/ET(A)R autocrine axis in promoting epithelial to mesenchymal transition (EMT) in ovarian tumor cells, a key event in cancer metastasis, in which epithelial cells depolarize, disassemble cell-cell contacts, and adopt an invasive phenotype. Here, we examine the potential role of ET-1 in regulating cell morphology and behavior and epithelial and mesenchymal proteins employing an in vitro 3-D culture system. We found that in 3-D serum-free collagen I gel cultures, HEY and OVCA 433 ovarian carcinoma cells undergo fibroblast-like morphologic changes between 3 and 5 days of ET-1 treatment. In these cells, ET-1 induces loss of adherens and tight-junction protein expression, E-cadherin, beta-catenin, and zonula occludens-1, and gain of N-cadherin and vimentin expression. These results confirm the ability of ET-1 to promote EMT, a metastable process involving sustained loss of epithelial markers and gain of mesenchymal markers. Collectively, these findings provide evidence of a critical role for the ET-1/ET(A)R axis during distinct steps of ovarian carcinoma progression, thus underlining this axis as a potential target in the treatment of ovarian cancer.

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 (ET(A)R) 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 beta1-integrin and growth factor receptors. In this study, we investigate whether the signaling triggered by ET(A)R leading to an aggressive phenotype is mediated by an ILK-dependent mechanism. In HEY and OVCA 433 ovarian carcinoma cell lines, activation of ET(A)R by ET-1 enhances the expression of alpha2beta1 and alpha3beta1 integrins. ILK activity increases as ovarian cancer cells adhere to type I collagen through beta1 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-3beta. The blockade of ET-1/ET(A)R-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 ET(A)R antagonist, suppresses ILK expression, Akt and glycogen synthase kinase-3beta phosphorylation, and tumor growth. These data show that ILK functions as a downstream mediator of the ET-1/ET(A)R axis to potentiate aggressive cellular behavior. Thus, the ILK-related signaling cascade can be efficiently targeted by pharmacologic blockade of ET(A)R.

cDNA-array profiling of melanomas and paired melanocyte cultures.

Three paired (from the same donor) sets of melanoma cells and normal melanocytes, established as early-passage cultures from metastatic lesions and the uninvolved skin of three patients, were comparatively cDNA profiled by macroarrays (approximately 1,200 genes) and reverse transcription (RT)-PCR. While 145 gene products were significantly (at least twofold) upregulated or downregulated in at least 1 pair, and 23 were in at least 2 pairs, only 3 (the signal transducer and activator of transcription STAT2, collagen type VI, and CD9) were concordantly modulated (downregulation) in all 3 pairs. Array results were validated by RT-PCR on a small panel of surgically removed nevocellular nevi and metastatic melanoma lesions, and by immunohistochemistry on a large panel of benign and malignant lesions of the nevomelanocytic lineage. The three gene products were downregulated at different stages of melanoma progression. STAT2 was detectable in nevi (5/5) and most primary melanomas (11/12), but was lost in 10/15 metastatic lesions. Collagen type VI was expressed in nevi (5/5) and primary melanomas below a Breslow thickness of 1 mm (3/3), but was lost in 24/24 primary melanomas above this threshold, and in metastatic melanomas (10/10). The tetraspanin CD9 molecule was expressed in 18/18 nevi, but was lost in 20/28 primary melanomas (including thin lesions), and in 24/52 metastatic lesions. These data provide the proof of principle that cDNA profiling of paired melanocyte/melanoma cultures sorts out novel, early signatures of melanocyte transformation that could contribute to the clinical management of patients at high risk of metastatic disease.

Phenotypic and functional changes of human melanoma xenografts induced by DNA hypomethylation: immunotherapeutic implications.

Emerging in vitro evidence points to an immunomodulatory activity of DNA hypomethylating drugs in human malignancies. We investigated the potential of 5-aza-2'-deoxycytidine (5-AZA-CdR) to modulate the expression of cancer testis antigens (CTA) and of HLA class I antigens by melanoma xenografts, and the resulting modifications in immunogenicity of neoplastic cells. Three primary cultures of melanoma cells, selected for immune phenotype and growth rate, were grafted into BALB/c nu/nu mice that were injected intraperitoneally with different dose- and time-schedules of 5-AZA-CdR. Molecular analyses demonstrated a de novo long-lasting expression of the CTA MAGE-1, -2, -3, -4, -10, GAGE 1-6, NY-ESO-1, and the upregulation of MAGE-1, MAGE-3, and NY-ESO-1 levels in melanoma xenografts from 5-AZA-CdR-treated mice. Serological and biochemical analyses identified a de novo expression of NY-ESO-1 protein and a concomitant and persistent upregulation of HLA class I antigens and of HLA-A1 and -A2 alleles. Immunization of BALB/c mice with 5-AZA-CdR-treated melanoma cells generated high titer circulating anti-NY-ESO-1 antibodies. Altogether, the data obtained identify an immunomodulatory activity of 5-AZA-CdR in vivo and strongly suggest for its clinical use to design novel strategies of CTA-based chemo-immunotherapy for melanoma patients.

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.

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

PURPOSE AND EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.