Identification of c-Jun dependent genes downstream of acute MITF loss in the context of inflammatory signals in the human melanoma cell line Ma-Mel-15
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Michael Holzel
PROVIDER: E-GEOD-71886 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:We analyzed the transcriptional response of the human melanoma cell line Ma-Mel-15 either transfected with control siRNA (siNT = non-targeting siRNA) or transfected with siRNAs (pool of 4 active and independent siRNAs) directed against the melanocytic transcription factor and lineage oncogene MITF (Microphthalmia-associated transcription factor). The experiment was performed as biological duplicates and RNA was isolated 48 hours after siRNA transfection. We aimed to determine novel markers and pathways of melanoma cell plasticity. Total RNA was obtained from siRNA-treated Ma-Mel-15 melanoma cell lines and global gene expression profiling was done using the Illumina Human HT12 v4 platform.
2015-10-29 | E-GEOD-71881 | biostudies-arrayexpress
Project description:Ma-Mel-15 human melanoma cell cultures were transiently transfected (RNAiMax, Lipofectamin) with control siRNA, siRNA against MITF (pool of 4 siRNAs), siRNA against c-JUN (pool of 4 siRNAs) or combinations of siMITF and siJUN. Cells were then either treated with TNF-alpha (1000U/ml) for 24 hours or left untreated. The experiment was performed as biological duplicates. We aimed to determine how c-JUN cooperates with acute MITF-loss in human melanoma cells to increase inflammatory responsiveness and cell plasticity.
2015-10-29 | GSE71886 | GEO
Project description:We analyzed the transcriptional response of the human melanoma cell line MZ7 to TNF-alpha (24 hours) in a dose-dependent manner (TNF-alpha 10U/ml, 100U/ml, 1000U/ml) either transfected with control siRNA (siNT = non-targeting siRNA) or transfected with siRNAs (pool of 4 active and independent siRNAs) against the melanocytic transcription factor and lineage oncogene MITF. (Microphthalmia-associated transcription factor). The experiment was performed as biological duplicate. As MITF is critical for melanoma cell state control, we aimed to explore how MITF expression intersects with inflammation-induced plasticity pathways in melanoma. Total RNA was obtained from siRNA/TNF-treated MZ7 melanoma cell lines at various conditions and global gene expression profiling was done using the Illumina Human HT12 v4 platform.
2015-10-29 | E-GEOD-71798 | biostudies-arrayexpress
Project description:Little is known about genes that promote melanoma cell growth and proliferation. siRNAs may be used to address the role of individual genesin these processes. RNAi library screens were used in the past to gain a comprehensive overview of all genes involved in cell growth, proliferation, migration and other cellular processes. A large-scale loss-of-function screen for eight different melanoma cell lines was performed using a pooled lentiviral shRNA library (GeneNet Human 50K lentiviral shRNA Library,cat#SI206B-1, System Biosciences) to identify genes relevant for melanoma cell growth and proliferation. shRNAs that lead to cell death or reduced growth of transduced melanoma cells are negatively selected and thereby underrepresented in the final cellular shRNA pool and vice versa. The shRNAs of the shRNA library (3-5 per gene) have complementary sequences to probes on the custom Affymetrix microarray HG-U133Plus2 and were analysed using this array. Well-known melanoma cell lines SK-Mel-103, A375, SK-Mel-147, SK-Mel-19, SK-Mel-28, SK-Mel-29, SK-Mel-5, WM3523cln6 were transduced with the lentiviral shRNA library and grown for 10 days under puromycin selection (day 10), control cells of respective cell lines were transduced and frozen immediately after transduction and genomic integration of shRNAs (day 0). Totel DNA was extracted and genomically integrated shRNAs were hybridized to Affymetrix microarrays (HG-U133Plus2.0 array).
2014-01-24 | E-GEOD-46675 | biostudies-arrayexpress
Project description:Canonical Wnt signaling plays an important role in development and disease, regulating transcription of target genes and stabilizing many proteins phosphorylated by Glycogen Synthase Kinase 3 (GSK3). We observed that the MiT family of transcription factors, which includes the melanoma oncogene MITF and the lysosomal master regulator TFEB, had the highest phylogenetic conservation of three consecutive putative GSK3 phosphorylation sites in animal proteomes. This prompted us to examine the relationship between MITF, endolysosomal biogenesis and Wnt signaling. Here we report that MITF expression levels correlated with the expression of a large subset of lysosomal genes in melanoma cell lines. MITF expression in the Tetracycline-inducible C32 melanoma model caused a marked increase in vesicular structures, and increased expression of late endosomal proteins such as Rab7, LAMP1, and CD63. These late endosomes were not functional lysosomes as they were less active in proteolysis, yet were able to concentrate Axin1, phospho-LRP6, phospho-β-Catenin, and GSK3 in the presence of Wnt ligands. This relocalization significantly enhanced Wnt signaling by increasing the number of multivesicular bodies (MVBs) into which the Wnt signalosome/destruction complex becomes localized upon Wnt signaling. We also show that the MITF protein was stabilized by Wnt signaling, through the novel C-terminal GSK3 phosphorylations identified here. MITF stabilization caused an increase in MVB biosynthesis, which in turn increased Wnt signaling, generating a positive feed-back loop that may function during the proliferative stages of melanoma. The results underscore the importance of misregulated endolysosomal biogenesis in Wnt signaling and cancer. Expression of selected Lysosomal genes and CLEAR element plus MITF were compared in 51 melanoma cell lines to a mixed reference pool containing equal amounts of 47 melanoma cell lines.
2014-12-24 | E-GEOD-64497 | biostudies-arrayexpress
Project description:Efficient processing of target antigens by the ubiquitin-proteasome-system (UPS) is essential for treatment of cancers by T cell therapies. However, immune escape due to impaired expression of IFN-γ-inducible components of the antigen presentation machinery and consequent inefficient processing of HLA-dependent tumor epitopes can be one important reason for failure of such therapies. Here, we show that repeated short-term co-cultures of Melan-A/MART-1 tumor antigen-expressing melanoma cells with Melan-A/MART-1 (26-35)-specific CTL led to the generation of clones resistant to CTL-mediated cell death. To determine which of the UPS components and its associated pathways was responsible for CTL escape; three UKRV-Mel-15a clones were subjected to microarray gene expression analysis. Three UKRV-Mel-15a-derived melanoma clones were isolated following three repeated short-term exposures to Melan-A/MART (26-35) CTL and harvested for RNA extraction and hybridization on Affymetrix microarrays.
2015-12-12 | E-GEOD-75929 | biostudies-arrayexpress
Project description:We analyzed the transcriptional response of the human melanoma cell line Ma-Mel-15 either transfected with control siRNA (siNT = non-targeting siRNA) or transfected with siRNAs (pool of 4 active and independent siRNAs) directed against the melanocytic transcription factor and lineage oncogene MITF (Microphthalmia-associated transcription factor). The experiment was performed as biological duplicates and RNA was isolated 48 hours after siRNA transfection. We aimed to determine novel markers and pathways of melanoma cell plasticity.
2015-10-29 | GSE71881 | GEO
Project description:The transcription factors PAX3 and MITF are required for the development of the neural crest and melanocyte lineage, and both proteins play important roles in melanoma cell growth and survival. PAX3 transcriptionally activates MITF expression during neural crest development, but the relationship between these transcription factors during melanocyte development and in melanoma cells is currently poorly understood. This study aimed to further our understanding of the interaction between transcriptional networks controlled by PAX3 and MITF by assessing the effect of siRNA-mediated knockdown of PAX3 and MITF in metastatic melanoma cell lines. The goals of this study were to determine (i) if PAX3 is required for maintaining expression of MITF in melanoma and melanocyte cell lines; (ii) whether PAX3 and MITF independently, or redundantly, influence growth and survival in melanoma cell lines; and (iii) to investigate the respective roles of PAX3 and MITF expression in melanoma cell differentiation. Microarrays were used to measure global changes in transcript expression in response to siRNA-mediated knockdown of PAX3 or MITF compared to non-targeting controls in two metastatic melanoma cells lines. RNA was isolated from two different metastatic melanoma cell lines 30 hours after one of four different treaments: (i) transfection with siRNA targeting PAX3; or (ii) transfection with siRNA targeting MITF; or (iii) or transfection with siRNA targeting luciferase (non-targeting negative control); or (iv) treatment with media only (control). Therefore, eight samples were used for gene expression profiling by using GeneChip arrays, with one replicate per cell line per treatment.
2010-01-01 | E-GEOD-16249 | biostudies-arrayexpress
Project description:We previously identified the induction of growth arrest with phenotypic characteristics of senescence in melanoma cell lines sensitive to diterpene esters, indicating a therapeutic potential. Here we compared the cytostatic effects of two diterpene esters namely TPA (12-O-tetradecanoylphorbol-13-acetate) and PEP008 (20-O-acetyl-ingenol-3-angelate) in sensitive and resistant cell lines derived from melanoma, breast cancer and colon cancer. We showed the diterpene esters to induce senescence-like growth arrest in the sensitive cells at 100-1000 ng/ml. Use of the pan-PKC inhibitor bisindolylmaleimide-l demonstrated that activation of PKC was required for growth arrest. Full genome expression profiling revealed that pivotal genes involved in DNA synthesis and cell cycle control were down-regulated by treatment in all three sensitive solid tumor models. At the protein level, prolonged down-regulation of E2F-1 and proliferating cell nuclear antigen (PCNA), sustained expression of p21WAF1/CIP1 and dephosphorylation of retinoblastoma (Rb) occurred in the sensitive cells. Although activation of extracellular signal-related kinase (ERK) 1/2 by the diterpene esters occurred in both sensitive and resistant cell lines, the HRASLS3 type II tumor suppressor, which appears to have a role in MAPK pathway suppression, was constitutively elevated in the resistant cell lines compared to their sensitive counterparts. Together, these results demonstrate the ability of the PKC activating drugs TPA and PEP008 to induce growth arrest with characteristics of senescence in solid tumor cell lines derived from a variety of tissue types through a similar mechanism. PKC-activating diterpene esters may therefore have therapeutic potential in a range of solid tumors. Experiment Overall Design: We analyzed the transcriptional profiles of the diterpene ester sensitive cell lines MCF7, COLO-205 and SK-MEL-5 following treatment with PEP008 using full genome expression profiling (Affymetrix, U133 Plus 2.0). Cells were treated for 24 h and 24 h plus 72 h recovery with 1000 ng/ml of the drug, before harvesting RNA for analysis. From the cell growth assays, all three cell lines demonstrated permanent growth arrest with diterpene ester treatments at the 1000 ng/ml dose. Mock controls were treated with solvent alone for 24 h. SK-MEL-5 cells were also treated with 1000 ng/ml TPA for 24 h.
2008-06-16 | E-GEOD-8742 | biostudies-arrayexpress
Project description:MicroRNA (miRNA) expression profiling identified miR-638 as one of the most significantly overexpressed miRNAs in metastatic lesions compared with primary melanomas. miR-638 enhanced the tumourigenic properties of melanoma cells in vitro and lung colonization in vivo. mRNA expression profiling of miR-638 and antagomir-transduced cells identified new candidate genes as miR-638 targets, the majority of which is involved in p53-mediated apoptosis regulation. miR-638 depletion stimulated expression of p53 and its downstream target genes and induced apoptosis and autophagy in melanoma cells. miR-638 promoter analysis revealed transcription factor associated protein 2-? (TFAP2A) as a direct negative regulator of miR-638. Further analyses provided strong evidence for a double negative regulatory feedback loop between miR-638 and TFAP2A. Taken together, miR-638 may support melanoma progression by suppressing p53-mediated apoptosis pathways and by targeting the transcriptional repressor TFAP2A. Whole genome cDNA microarray (Illumina Human HT-12 v4 Expression BeadChip Kit, San Diego, CA 92122 USA) analyses were performed in duplicates using RNA extracted from SK-Mel-147 cells transfected with a non-targeting control, miR-638 or antagomiR-638.
2014-01-30 | E-GEOD-54493 | biostudies-arrayexpress