a-192621 and Melanoma

The G-protein-coupled receptor, endothelin receptor B (EDNRB), is an important regulator of melanocyte survival and proliferation. It acts by stimulating downstream heterotrimeric G proteins, such as Gα

Topics: Animals; Carcinogenesis; Dermis; Endothelins; GTP-Binding Protein alpha Subunits, Gq-G11; Lung Neoplasms; Melanocytes; Melanoma; Meningeal Neoplasms; Mice, Knockout; Phenotype; Pyrrolidines; Receptor, Endothelin B; RNA, Untranslated; Signal Transduction; Skin Neoplasms; Uveal Neoplasms

Metastatic spread of melanoma to the central nervous system (CNS) is a common and devastating manifestation of disease progression, which, despite its clinical importance, remains poorly understood with respect to underlying molecular mechanisms. Using a recently developed preclinical model of spontaneous melanoma CNS metastasis, we have identified alterations in expression of endothelin receptor B (EDNRB) as a potential factor that influences brain metastatic potential. Induced overexpression of this gene mediated enhanced overall metastatic disease, and resulted in an increased incidence of spontaneous CNS metastases. In contrast, the overexpression of other highlighted genes, such as BCL2A1, did not affect the incidence of CNS metastases but nevertheless appears to facilitate intracranial tumor growth. The prometastatic effect in the CNS associated with EDNRB appears to be mediated by the interaction with its ligands resulting in enhanced tumor cell proliferation and thus intracranial melanoma growth. That EDNRB contributes to melanoma metastasis is underscored by the fact that its therapeutic inhibition by the EDNRB-specific inhibitor A192621 translated into improved outcomes when treating mice with either visceral metastases or intracranial tumors. The identification of an influential role of EDNRB in CNS melanoma spontaneous metastasis may provide both a target for therapeutic intervention as well as a potential prognostic marker for patients having an increased predisposition for incidence of CNS melanoma metastases.

Topics: Animals; Cell Line, Tumor; Central Nervous System Neoplasms; Endothelin B Receptor Antagonists; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Mice; Mice, SCID; Minor Histocompatibility Antigens; Oligonucleotide Array Sequence Analysis; Prognosis; Proto-Oncogene Proteins c-bcl-2; Pyrrolidines; Receptor, Endothelin B; Reverse Transcriptase Polymerase Chain Reaction; Survival Analysis; Tumor Burden; Xenograft Model Antitumor Assays

Endothelin receptor antagonists inhibit the progression of many cancers, but research into their influence on glioma has been limited.. We treated glioma cell lines, LN-229 and SW1088, and melanoma cell lines, A375 and WM35, with two endothelin receptor type B (ETRB)-specific antagonists, A-192621 and BQ788, and quantified viable cells by the capacity of their intracellular esterases to convert non-fluorescent calcein AM into green-fluorescent calcein. We assessed cell proliferation by labeling cells with carboxyfluorescein diacetate succinimidyl ester and quantifying the fluorescence by FACS analysis. We also examined the cell cycle status using BrdU/propidium iodide double staining and FACS analysis. We evaluated changes in gene expression by microarray analysis following treatment with A-192621 in glioma cells. We examined the role of ETRB by reducing its expression level using small interfering RNA (siRNA).. We report that two ETRB-specific antagonists, A-192621 and BQ788, reduce the number of viable cells in two glioma cell lines in a dose- and time-dependent manner. We describe similar results for two melanoma cell lines. The more potent of the two antagonists, A-192621, decreases the mean number of cell divisions at least in part by inducing a G2/M arrest and apoptosis. Microarray analysis of the effects of A-192621 treatment reveals up-regulation of several DNA damage-inducible genes. These results were confirmed by real-time RT-PCR. Importantly, reducing expression of ETRB with siRNAs does not abrogate the effects of either A-192621 or BQ788 in glioma or melanoma cells. Furthermore, BQ123, an endothelin receptor type A (ETRA)-specific antagonist, has no effect on cell viability in any of these cell lines, indicating that the ETRB-independent effects on cell viability exhibited by A-192621 and BQ788 are not a result of ETRA inhibition.. While ETRB antagonists reduce the viability of glioma cells in vitro, it appears unlikely that this effect is mediated by ETRB inhibition or cross-reaction with ETRA. Instead, we present evidence that A-192621 affects glioma and melanoma viability by activating stress/DNA damage response pathways, which leads to cell cycle arrest and apoptosis. This is the first evidence linking ETRB antagonist treatment to enhanced expression of DNA damage-inducible genes.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Ciprofloxacin; DNA Damage; Endothelin B Receptor Antagonists; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioma; Humans; Isoquinolines; Melanoma; Oligonucleotide Array Sequence Analysis; Pyrroles; Pyrrolidines; Receptor, Endothelin A; Receptor, Endothelin B; RNA, Small Interfering

Phenotypic and genotypic analyses of cutaneous melanoma have identified the endothelin B receptor (ET(B)R) as tumor progression marker, thus representing a potential therapeutic target. Here, we demonstrate that activation of ET(B)R 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 ET(B)R pathway increases alpha(v)beta(3) and alpha(2)beta(1) integrin expression and matrix metalloproteinase (MMP)-2 and MMP-9, membrane type-1-MMP activation, and tissue inhibitor MMP-2 secretion. The ET(B)R 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 ET(B)R antagonist, significantly inhibits melanoma growth in nude mice. These findings demonstrate that ET-1 and ET-3 through ET(B)R activation trigger signaling pathways involved in events associated with disruption of normal host-tumor interactions and progression of cutaneous melanoma. Pharmacological interruption of ET(B)R signaling may represent a novel therapeutic strategy in the treatment of this malignancy.

Topics: Animals; Cadherins; Cell Communication; Cell Line, Tumor; Endothelin B Receptor Antagonists; Endothelin-1; Endothelin-3; Enzyme Activation; Female; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinases, Membrane-Associated; Melanoma; Metalloendopeptidases; Mice; Mice, Nude; Mitogen-Activated Protein Kinases; Pyrrolidines; Receptor, Endothelin B; Signal Transduction