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SiRNA-mediated knockdown of MITF and PAX3 in metastatic melanoma cell lines

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE16249 | GEO | 2010/01/02

SECONDARY ACCESSION(S): PRJNA115189

REPOSITORIES: GEO

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siRNA-mediated knockdown of MITF and PAX3 in metastatic melanoma cell lines

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

DNA Promoter Hypermethylation of Melanocyte Lineage Genes Determines Melanoma Phenotype

Project description:Cellular stress contributes to the capacity of melanoma cells to undergo phenotype switching into highly migratory and drug tolerant dedifferentiated states. Such dedifferentiated melanoma cell states are marked by loss of melanocyte specific gene expression and increase of mesenchymal markers. Two crucial transcription factors, MITF and SOX10, important in melanoma development and progression have been implicated in this process. In this study we describe that loss of MITF is associated with a distinct transcriptional program, MITF promoter hypermethylation and poor patient survival in metastatic melanoma. From a comprehensive collection of melanoma cell lines, we observed that MITF methylated cultures were subdivided in two distinct subtypes. Examining mRNA levels of neural crest associated genes we found that one subtype had lost the expression of several lineage genes including SOX10. Intriguingly, SOX10 loss was associated with SOX10 gene promoter hypermethylation and distinct phenotypic and metastatic properties. Depletion of SOX10 in MITF methylated melanoma cells using CRISPR/Cas9 confirmed these findings. In conclusion, this study describes the significance of melanoma state and the underlying functional properties explaining the aggressiveness of such states.

2022-12-08 | GSE211906 | GEO

Homo sapiens

Project description:siRNA-mediated knockdown of MITF and PAX3 in metastatic melanoma cell lines

| PRJNA115189 | ENA

MITF deficiency and oncogenic GNAQ each promote proliferation programs in zebrafish melanocyte lineage cells

Project description:Uveal melanoma (UM) is the most common primary malignancy of the eye in adults, but lacks any FDA-approved therapy for the deadly metastatic disease. Thus, there is great need to dissect the driving mechanisms for UM, and develop strategies to evaluate potential therapeutics. Using a zebrafish model, we previously identified MITF, the master melanocyte transcription factor, as a tumor suppressor in GNAQQ209L-driven UM. Here, we show that zebrafish mitfa-deficient GNAQQ209L-driven tumors significantly up-regulate neural crest markers, and higher expression of a melanoma-associated neural crest signature correlates with poor UM patient survival. We further determined how the mitfa-null state, as well as expression of GNAQQ209L, YAPS127A;S381A, or BRAFV600E oncogenes, impacts melanocyte lineage cells before they acquire the transformed state. Specifically, examination 5 days post-fertilization showed that mitfa-deficiency is sufficient to upregulate pigment progenitor and neural crest markers, while GNAQQ209L expression promotes a proliferative phenotype that is further enhanced by YAPS127A;S381A co-expression. Finally, we show that this oncogene-induced proliferative phenotype can be used to screen chemical inhibitors for their efficacy against the UM pathway. Overall, this study establishes that a neural crest signature correlates with poor UM survival, and describes an in vivo assay for pre-clinical trials of potential UM therapeutics.

2022-07-25 | GSE197927 | GEO

Chromatin remodellers Brg1 and Bptf are required for normal gene expression and progression of oncogenic Braf-driven mouse melanoma [ChIP-seq]

Project description:Somatic oncogenic mutation of BRAF coupled with inactivation of PTEN constitute a frequent combination of genomic alterations driving development of human melanoma. Mice genetically engineered to conditionally express oncogenic BrafV600E and inactivate Pten in melanocytes following tamoxifen treatment rapidly develop melanoma. While early stage melanomas comprised melanin-pigmented Mitf and Dct-expressing cells, expression of these and other melanocyte identity genes was lost in later stage tumours that showed histological and molecular characteristics of de-differentiated neural crest type cells. Melanocyte identity genes displayed loss of active chromatin marks and RNA polymerase II and gain of heterochromatin marks indicating epigenetic reprogramming during tumour progression. Nevertheless, late stage tumour cells grown in culture re-expressed Mitf and melanocyte markers and Mitf together with Sox10 co-regulated a large number of genes essential for their growth. In this melanoma model, somatic inactivation that the catalytic Brg1 (Smarca4) subunit of the SWI/SNF complex and the scaffolding Bptf subunit of the NuRF delayed tumour formation and deregulated large and overlapping gene expression programs essential for normal tumour cell growth. Moreover, we show that Brg1 and Bptf co-regulated many genes together with Mitf and Sox10. Together these transcription factors and chromatin remodelling complexes orchestrate essential gene expression programs in mouse melanoma cells

2019-04-17 | GSE100998 | GEO

Clinical relevance of SKP2 alterations in metastatic melanoma

Project description:Melanoma cell lines were genotyped to evaluate copy number differences between nodular melanoma (NM) and superficial spreading melanoma (SSM). Cell lines were also evaluated for copy number alterations in the SKP2/p27 axis. Affymetrix SNP arrays were performed according to manufacturer's instructions using DNA extracted from 18 melanoma cell lines and 4 melanocyte controls. Affymetrix SNP6.0 Array data for melanoma cell lines Copy number analysis of Affymetrix SNP 6.0 arrays was performed on 18 melanoma cell lines including 2 primary superficial spreading melanoma, 2 primary nodular melanoma, 2 metastatic nodular melanoma, and 12 metastatic cell lines. Four melanocyte control lines were also evaluated including 2 immortalized melanocyte cell lines (Hermes 1 and 2B) and 2 normal melanocyte lines cultured from neonatal foreskin (HEM-N and HEM-LP) that were used to construct the baseline for copy number analysis.

2010-10-21 | E-GEOD-22305 | biostudies-arrayexpress

Genome-wide analysis of differential gene expression in human embryonic stem cell-derived central nervous system, neural crest, and progenitor populations over time

Project description:Melanocytes are pigment-producing cells of neural crest origin responsible for protecting the skin against UV-irradiation. Melanocyte dysfunction leads to pigmentation defects including albinism, vitiligo, and piebaldism and is a key feature of systemic pathologies such as Hermansky-Pudlak (HP) and Chediak-Higashi (CH) Syndromes. Pluripotent stem cell technology offers a novel approach for studying human melanocyte development and disease. Here we report that timed exposure to activators of WNT, BMP and EDN3 signaling triggers the sequential induction of neural crest and melanocyte precursor fates under dual-SMAD inhibition conditions. Using a SOX10::GFP hESC reporter line, we demonstrate that the temporal onset of WNT activation is particularly critical for human neural crest induction. Surprisingly, suppression of BMP signaling does reduce neural crest yield. Subsequent differentiation of hESC-derived melanocyte precursors under defined conditions yields pure populations of pigmented cells matching the molecular and functional properties of adult melanocytes. Melanocytes from patient-specific iPSCs faithfully reproduce the ultrastructural features of the HP- and CH-specific pigmentation defects with minimal variability across lines. Our data define a highly specific requirement for WNT signaling during neural crest induction and enable the generation of pure populations of hiPSC-derived melanocytes for faithful modeling of human pigmentation disorders. Total RNA obtained from a timecourse of Dual SMAD Inhibition (DSi), Neural Crest (NC), and Melanocyte (BE) differentiation of human embryonic stem cells in triplicate.

2013-06-01 | E-GEOD-45223 | biostudies-arrayexpress

Deep sequencing analysis of microRNA expression in human melanocyte and melanoma cell lines

Project description:We investigated the miRNAome in human melanocyte and melanoma cell lines using high-throughput RNA sequencing. We identified a group of dysregulated miRNAs by comparing the miRNA expression profiles among melanoma cell lines. Target genes of these miRNAs participate in functions associated with the cell cycle and apoptosis. Gene networks were built to investigate the interactions of genes during melanoma progression. We identified that the key genes that regulate melanoma cell proliferation were regulated by miRNAs. Our findings provide further knowledge regarding the mechanisms of melanoma development. miRNA profiles of melanocyte (HEMn-LP), low metastatic melanoma (A375) and high metastatic melanoma (A2058) cell line were generated using Illumina GA

2015-07-13 | E-GEOD-66823 | biostudies-arrayexpress

Modulation of the transcriptional response to TNF-alpha in the human melanoma cell line MZ7 by MITF

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

Analysis of p53 target genes in melanoma part 1

Project description:Although mutations in p53 are infrequent in human melanoma, its function is abnormal. In this study, whole genome bead arrays were used to examine the expression of p53 target genes in extracts from 82 metastatic melanoma samples, compared to extracts derived from diploid human melanocytes, to provide a global assessment of aberrant p53 function. Total RNA extracted from 82 tumour samples and 8 melanocyte cell lines was analysed. Metastatic melanomas were compared to melanocyte cell lines.

2011-12-01 | E-GEOD-29359 | biostudies-arrayexpress

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