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Genetic and molecular characterization of uveal melanoma cell lines.

ABSTRACT: The recent identification of frequent activating mutations in GNAQ or GNA11 in uveal melanoma provides an opportunity to better understand the pathogenesis of this melanoma subtype and to develop rational therapeutics to target the cellular effects mediated by these mutations. Cell lines from uveal melanoma tumors are an essential tool for these types of analyses. We report the mutation status of relevant melanoma genes, expression levels of proteins of interest, and DNA fingerprinting of a panel of uveal melanoma cell lines used in the research community.

SUBMITTER: Griewank KG

PROVIDER: S-EPMC3288394 | biostudies-other | 2012 Mar

REPOSITORIES: biostudies-other

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Genetic and molecular characterization of uveal melanoma cell lines.

Griewank K G KG Yu X X Khalili J J Sozen M M MM Stempke-Hale K K Bernatchez C C Wardell S S Bastian B C BC Woodman S E SE

Pigment cell & melanoma research 20120202 2

The recent identification of frequent activating mutations in GNAQ or GNA11 in uveal melanoma provides an opportunity to better understand the pathogenesis of this melanoma subtype and to develop rational therapeutics to target the cellular effects mediated by these mutations. Cell lines from uveal melanoma tumors are an essential tool for these types of analyses. We report the mutation status of relevant melanoma genes, expression levels of proteins of interest, and DNA fingerprinting of a pane ...[more]

PMID: 22236444

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Characterization of Uveal Melanoma Cell Lines and Primary Tumor Samples in 3D Culture.

Project description:PurposeUveal melanoma (UM) typically spreads to the liver, where it is incurable, as there are limited therapeutic interventions available. This study aimed to standardize laboratory methods for generating three-dimensional (3D) spheroids using UM cell lines and primary UM (PUM) samples for use in drug screening.MethodsSix UM cell lines and nine PUM, of differing genetic characteristics were cultured in two dimensions (2D) and three dimensions. 3D spheroid formation and growth were time monitored, and ImageJ software was used to calculate cross-sectional areas. PUM spheroids underwent immunohistochemistry for melanoma markers, nuclear BAP1, and cell proliferation. Chromosomal alterations in patient UM biopsies were compared with the corresponding 3D spheroid. In vitro drug assays testing doxorubicin and selumetinib assessed drug penetration and toxicity after 48 hours using imaging and the CellTiter-Glo 3D Cell Viability Assay.ResultsAll six UM cell lines formed spheroids of varying sizes and compactness; six of the nine PUM samples (67%) also formed spheroids, composed of MelanA+ proliferating melanocytes and admixed macrophages. PUM spheroids were genetically identical to the original sampled tumor. In vitro drug assays showed varying penetrations into UM cell line spheroids, with doxorubicin passing into the spheroid core and selumetinib having an effect largely on peripheral cells. Both drugs caused a dose-dependent reduction in viability of 3D spheroid cells.ConclusionsUM cell lines and PUM samples can successfully generate uniform 3D spheroids. PUM spheroids retain histological and genetic characteristics of the primary tumor. 3D spheroids are an important system for use in future high-throughput drug testing.Translational relevanceThe use of 3D spheroids allows early-phase drug screening and is an important first step toward treatment personalization for UM patients.

| S-EPMC7414609 | biostudies-literature

Primary uveal melanoma cells, normal uveal melanocytes, neural crest stem cells, embryonic stem cells and uveal melanoma cell lines

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

| S-ECPF-GEOD-44299 | biostudies-other

Molecular pathology of uveal melanoma.

Project description:Like other cancers, uveal melanomas (UM) are characterised by an uncontrolled, clonal, cellular proliferation, occurring as a result of numerous genetic, and epigenetic aberrations. Signalling pathways known to be disrupted in UM include: (1) the retinoblastoma pathway, probably as a result of cyclin D1 overexpression; p53 signalling, possibly as a consequence of MDM2 overexpression; and the P13K/AKT and mitogen-activated protein kinase/extracellular signal-related kinase pathway pathways that are disturbed as a result of PTEN and GNAQ/11 mutations, respectively. Characteristic chromosomal abnormalities are common and include 6p gain, associated with a good prognosis, as well as 1p loss, 3 loss, and 8q gain, which correlate with high mortality. These are identified by techniques such as fluorescence in situ hybridisation, comparative genomic hybridisation, microsatellite analysis, multiplex ligation-dependent probe amplification, and single-nucleotide polymorphisms. UM can also be categorised by their gene expression profiles as class 1 or class 2, the latter correlating with poor survival, as do BRCA1-associated protein-1 (BAP1) inactivating mutations. Genetic testing of UM has enhanced prognostication, especially when results are integrated with histological and clinical data. The identification of abnormal signalling pathways, genes and proteins in UM opens the way for target-based therapies, improving prospects for conserving vision and prolonging life.

| S-EPMC3574255 | biostudies-other

Mass Spectrometry-Based Profiling of Histone Post-Translational Modifications in Uveal Melanoma Tissues, Human Melanocytes, and Uveal Melanoma Cell Lines - A Pilot Study.

Project description:PurposeEpigenetic alterations in uveal melanoma (UM) are still neither well characterized, nor understood. In this pilot study, we sought to provide a deeper insight into the possible role of epigenetic alterations in the pathogenesis of UM and their potential prognostic relevance. To this aim, we comprehensively profiled histone post-translational modifications (PTMs), which represent epigenetic features regulating chromatin accessibility and gene transcription, in UM formalin-fixed paraffin-embedded (FFPE) tissues, control tissues, UM cell lines, and healthy melanocytes.MethodsFFPE tissues of UM (n = 24), normal choroid (n = 4), human UM cell lines (n = 7), skin melanocytes (n = 6), and uveal melanocytes (n = 2) were analyzed through a quantitative liquid chromatography-mass spectrometry (LC-MS) approach.ResultsHierarchical clustering showed a clear separation with several histone PTMs that changed significantly in a tumor compared to normal samples, in both tissues and cell lines. In addition, several acetylations and H4K20me1 showed lower levels in BAP1 mutant tumors. Some of these changes were also observed when we compared GNA11 mutant tumors with GNAQ tumors. The epigenetic profiling of cell lines revealed that the UM cell lines MP65 and UPMM1 have a histone PTM pattern closer to the primary tissues than the other cell lines analyzed.ConclusionsOur results suggest the existence of different histone PTM patterns that may be important for diagnosis and prognosis in UM. However, further analyses are needed to confirm these findings in a larger cohort. The epigenetic characterization of a panel of UM cell lines suggested which cellular models are more suitable for epigenetic investigations.

| S-EPMC10868634 | biostudies-literature

Fucoidan Does Not Exert Anti-Tumorigenic Effects on Uveal Melanoma Cell Lines.

Project description:The polysaccharide fucoidan is widely investigated as an anti-cancer agent. Here, we tested the effect of fucoidan on uveal melanoma cell lines.The effect of 100 µM fucoidan was investigated on five cell lines (92.1, Mel270 OMM1, OMM2.3, OMM2.5) and of 1 µg/mL-1 mg/mL fucoidan in two cell lines (OMM1, OMM2.3). Cell proliferation and viability were investigated with a WST-1 assay, migration in a wound healing (scratch) assay. Vascular Endothelial Growth Factor (VEGF) was measured in ELISA. Angiogenesis was evaluated in co-cultures with endothelial cells. Cell toxicity was induced by hydrogen-peroxide. Protein expression (Akt, ERK1/2, Bcl-2, Bax) was investigated in Western blot.Fucoidan increased proliferation in two and reduced it in one cell line. Migration was reduced in three cell lines. The effect of fucoidan on VEGF was cell type and concentration dependent. In endothelial co-culture with 92.1, fucoidan significantly increased tubular structures. Moreover, fucoidan significantly protected all tested uveal melanoma cell lines from hydrogen-peroxide induced cell death. Under oxidative stress, fucoidan did not alter the expression of Bcl-2, Bax or ERK1/2, while inducing Akt expression in 92.1 cells but not in any other cell line.Fucoidan did not show anti-tumorigenic effects but displayed protective and pro-angiogenic properties, rendering fucoidan unsuitable as a potential new drug for the treatment of uveal melanoma.

| S-EPMC5532635 | biostudies-other

Inflammation-induced chemokine expression in uveal melanoma cell lines stimulates monocyte chemotaxis

Project description:Purpose: Uveal melanoma (UM) is the most common primary intraocular tumor in adults and the presence of infiltrating leucocytes is associated with a poor prognosis. Little is known how infiltrating leucocytes influence the tumor cells. The purpose of this study was to investigate the effect of activated T cells on the expression of chemotactic cytokines in UM cells. Furthermore, we examined the ability of stimulated UM cells to attract monocytes. Methods: We used an in vitro co-culture system in which UM cell lines and T cells were cultured together, but separated by a membrane. UM gene expression was quantified using a microarray. Protein expression in the supernatant was quantified with ELISA or cytometric bead array. For the monocyte migration assay, a trans-well plate was used. Results: Gene-expression analysis of UM cell lines showed that co-culture with activated T cells resulted in an upregulation of chemokines such as CXCL8, CXCL9, CXCL10, CXCL11, CCL2, CCL5, vascular endothelial growth factor (VEGF), intracellular adhesion molecule 1 (ICAM1) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The upregulation of these molecules was confirmed at the protein level. This increase of chemokines coincided with an increased chemotactic capacity of the supernatant towards monocytes. Conclusions: Cytokines derived from activated T cells shifted the UM cell-transcriptome towards a more inflammatory state, including upregulation of several chemokines, which led to an increased migration of monocytes. Therefore, UM cells might actively participate in generating a tumor- promoting inflammatory microenvironment. One replicate for each cell lines (92.1, Mel 270 and Mel 290) was analyzed before and after co-culture with activated T cells.

| S-ECPF-GEOD-55983 | biostudies-other

Functional characterization of uveal melanoma oncogenes.

Project description:Uveal melanoma (UM) is a currently untreatable form of melanoma with a 50% mortality rate. Characterization of the essential signaling pathways driving this cancer is critical to develop target therapies. Activating mutations in the Gαq signaling pathway at the level of GNAQ, GNA11, or rarely CYSLTR2 or PLCβ4 are considered alterations driving proliferation in UM and several other neoplastic disorders. Here, we systematically examined the oncogenic signaling output of various mutations recurrently identified in human tumors. We demonstrate that CYSLTR2 → GNAQ/11 → PLCβ act in a linear signaling cascade that, via protein kinase C (PKC), activates in parallel the MAP-kinase and FAK/Yes-associated protein pathways. Using genetic ablation and pharmacological inhibition, we show that the PKC/RasGRP3/MAPK signaling branch is the essential component that drives the proliferation of UM. Only inhibition of the MAPK branch but not the FAK branch synergizes with inhibition of the proximal cascade, providing a blueprint for combination therapy. All oncogenic signaling could be extinguished by the novel GNAQ/11 inhibitor YM-254890, in all UM cells with driver mutation in the Gαq subunit or the upstream receptor. Our findings highlight the GNAQ/11 → PLCβ → PKC → MAPK pathway as the central signaling axis to be suppressed pharmacologically to treat for neoplastic disorders with Gαq pathway mutations.

| S-EPMC7856047 | biostudies-literature

YAP Activity is Not Associated with Survival of Uveal Melanoma Patients and Cell Lines.

Project description:Recent experimental studies have demonstrated an essential role for the Hippo-Yes-associated protein (YAP) pathway in GNAQ/GNA11-induced tumorigenesis in uveal melanoma (UM). However, the association between YAP activity and clinical outcomes remains elusive. We investigated possible associations between YAP activity and clinicopathological features including survival outcomes in patients with UM using The Cancer Genome Atlas (TCGA) cohort and our local cohort. We estimated YAP activity by mRNA expression levels, Gene Set Variation Analysis (GSVA) for the TCGA cohort, and immunohistochemical YAP staining for the local cohort. In the TCGA cohort, most clinicopathological features including tumor stage, mitotic counts, mutation of genes, and tumor sizes did not significantly differ between low and high YAP activity groups. In the local cohort, YAP nuclear-positive staining was observed in 30 (42%) of 72 patients with primary UM. UM-specific survival was not significantly different between tumors with low and high YAP activities. Unlike mesothelioma cells harboring a mutation of negative regulators of YAP, the survival of multiple UM cell lines was not significantly reduced by YAP/TAZ depletion. Our results suggest that the effect of YAP on development, growth, and invasion of UM in actual patients is less than previously demonstrated in experimental studies.

| S-EPMC7148330 | biostudies-literature

Establishment of novel cell lines recapitulating the genetic landscape of uveal melanoma and preclinical validation of mTOR as a therapeutic target.

Project description:Uveal melanoma (UM) is the most common primary tumor of the eye in adults. There is no standard adjuvant treatment to prevent metastasis and no effective therapy in the metastatic setting. We have established a unique panel of 7 UM cell lines from either patient's tumors or patient-derived tumor xenografts (PDXs). This panel recapitulates the molecular landscape of the disease in terms of genetic alterations and mutations. All the cell lines display GNAQ or GNA11 activating mutations, and importantly four of them display BAP1 (BRCA1 associated protein-1) deficiency, a hallmark of aggressive disease. The mTOR pathway was shown to be activated in most of the cell lines independent of AKT signaling. mTOR inhibitor Everolimus reduced the viability of UM cell lines and significantly delayed tumor growth in 4 PDXs. Our data suggest that mTOR inhibition with Everolimus, possibly in combination with other agents, may be considered as a therapeutic option for the management of uveal melanoma.

| S-EPMC5528590 | biostudies-literature

Genome-wide gene expression analysis of primary uveal melanoma cells, normal uveal melanocytes, neural crest stem cells, embryonic stem cells and uveal melanoma cell lines MUM2B and OCM1A

Project description:Gene expression in primary uveal melanoma cells and normal cell controls The HumanHT-12 v3 gene expression microarray (Illumina) was used to analyze RNA isolated from snap-frozen primary uveal melanoma tissue isolated from enucleated eyes and from normal cell controls.

| S-ECPF-GEOD-44295 | biostudies-other

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