Comparative gene expression profiles of immune inhibitory and non-inhibitory melanoma cell lines
ABSTRACT: Dysfunction in type I interferon (IFN) signaling occurs in patients with stage II or more advanced cancer. After screening the effects of a panel of 12 melanoma cell lines on PBMCs of healthy volunteers of IFNalpha signal pathway, two groups of melanoma cell lines could be identified one with stronger suppression (low pSTAT-1 group) than the other (high pSTAT-1 group). Comparative global gene expression between two groups identified 6771 differential expression genes. This gene list indicated down regulation of IFNalpha signal in immune suppressive melanoma cells. To evaluate this gene list for predictive power on IFNalpha signal modulatory function, we analyzed gene expression 41 independent melanoma cell lines and heat map clusters these cell lines into two groups, one with strong immune suppressive function and other with less effect. Fifty-three melanoma cell lines were analyzed with twelve technical repeats.
Project description:Dysfunction in type I interferon (IFN) signaling occurs in patients with stage II or more advanced cancer. After screening the effects of a panel of 12 melanoma cell lines on PBMCs of healthy volunteers of IFNalpha signal pathway, two groups of melanoma cell lines could be identified one with stronger suppression (low pSTAT-1 group) than the other (high pSTAT-1 group). Comparative genomic hybridization (CGH) identified consistent amplification of 12q22-24 as a genomic marker for the immune suppressive melanoma cell lines. This region corresponded to higher transcription of the NOS1 gene located in that region in the low pSTAT1 group and NOS1 expression was identified as causal factor in melanoma induced immune suppression. Twelve melanoma cell lines were analyzed. Genomic DNA of the cell line sample was labeled with Cy5 and reference normal PBMC DNA was labeled with Cy3.
Project description:The present study deals with functional interactions of cutaneous and brain-metastasizing human melanoma cells with brain-derived molecules. In this study we employed the unique melanoma xenograft model developed by Izraely and described in Int J Cancer. 2011 Oct 25. doi: 10.1002/ijc.27324. The present study aims to determine if brain-derived soluble factors regulate malignancy-associated functions of cutaneous and brain-metastasizing melanoma cells and identify which functions are regulated by such factors. The working hypothesis of this study is that the interactions between the brain microenvironment and melanoma cells determine metastasis formation at this organ site. The aim of the study was to evaluate the contribution of such interactions to the formation of brain metastasis in nude mice xenografted with human melanoma cells. An insight into these interactions is an essential pre-requisite for the development of effective targeted therapy for melanoma brain metastasis. We assessed the effects of soluble factors present in supernatants of short-term cultures of normal mouse brain (referred here after as brain-derived soluble factors) on several characteristics linked to melanoma brain metastasis. It was found that brain-derived soluble factors affect differentially cutaneous and brain-metastasizing melanoma cells variants in-vitro. Such factors enhanced the viability of cutaneous melanoma cells but caused an S phase arrest followed by apoptosis of brain-metastasizing cells. Brain-derived soluble factors enhanced migration of melanoma cells metastasizing to the brain, but did not affect the migration of the cutaneous variants. Such factors up-regulated the expression of the chemokine receptor CCR4 in both cutaneous and brain metastasizing melanoma cells. It is not unlikely that CCR4 ligands expressed in the brain interact with the CCR4-expressing melanoma cells thereby directing them to the brain. Brain-derived soluble factors enhanced the transmigration, across human brain endothelial cells of cutaneous but not of brain metastasizing melanoma variants. This activity could promote the capacity of the cutaneous cells to metastasize to the brain. 4 Samples (arrays) were analyzed. There is 1 replicate for each variant and each treatment. We generated pairwise comparisons between cutaneous and brain metastatic variants of the same genetic background, using Partek Genomics Suite, in the three melanoma models. Genes with p≤5% and a fold-change difference of ≥2 or <-2 were selected.
Project description:63 melanoma cell lines hybridized to Affymetrix Hu133_Plus 2 oligo arrays. The aim of this study was to identify potential downstream targets of key oncogenes and TSGs in melanoma (including p14ARF, p16INK4A, BRAF etc). Publications relevant to this series include:; Johansson et al. Pigment Cell Res 2007. Experiment Overall Design: 63 melanoma cell lines hybridized to Affymetrix Hu133_Plus 2 oligo arrays. These cell lines were sequenced for key tumour suppressor genes (TSGs) and oncogenes known to be involved in melanoma development. The microarray data was then analysed together with a particular genotype status (see relevant publications) to identify genes that potentially act downstream of these oncogenes and TSGs to contribute to melanoma development. See publications for analysis methods.
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.
Project description:MicroRNAs (miRNAs) influence cancer development through post-transcriptional negative regulation of both tumor suppressors and oncogenes. We subjected melanoma cell lines, normal melanocytes, and keratinocytes to array based miRNA profiling, and identified several distinct miRNAs with differential expression. Specifically, miR-211 levels were depleted in all eight melanoma cell lines examined, and also in 23 of 30 distinct patient melanoma samples (graded as primary in situ, regional metastatic, distant metastatic and nodal metastatic). Putative target genes of miR-211 were identified, and their anticipated increased expression levels were confirmed in melanoma cell lines, which were reduced in two melanoma cell lines that artificially over-expressed miR-211. Four such target genes (TCF12, RAB22A, KCNMA1 and SLC37A3) were confirmed by a target cleavage assay. Stable over-expression of miR-211 in two melanoma cell lines caused significant growth inhibition and reduced invasiveness. The differential expression of miR-211 in a variety of melanoma cell lines and clinical samples, consistent inverse correlation between miR-211 and its target mRNA levels, and growth retardation and reduced invasiveness of melanoma cell lines by miR-211 are all consistent with the idea that the depletion of miR-211 is a key step in melanoma development and/or progression The 15 Samples in this submission represent gene-level expression profiling of isolated total RNA from WM1552C, WM1552+miRNA211, A375, A375+miRNA211 and melanocytes hybridized to Affymetrix exon ararys.
Project description:Differential gene expression analysis of parental and resistant sub-lines of melanoma cell lines treated or untreated with PLX4032 Using microarray we sought to obtain a genome-wide profile of differentially expressed genes in parental melanoma cell lines and resistant sub-lines in response to PLX4032 vs DMSO control treatment. Representative parental melanoma cell lines and resistant sub-lines were treated with PLX4032 at 1 uM or DMSO control for 6h. Total RNA was extracted and cDNAs were generated and hybridized onto GeneChip Human Gene 1.0 ST Arrays (Affymetrix).
Project description:Expression profiles of 17 melanoma cell lines were analysed to identify genes differentially expressed between cell lines harbouring wild-type or mutant p16INK4A. Relevant paper: Pavey et al. (2007). Note: all of these cell lines contained wild-type p14ARF, so that the transcriptional effects of p16INk4A could be determined without interference from p14ARF. Experiment Overall Design: The aim of this study was to identify genes which are transcriptional targets of p16INK4A in melanoma.
Project description:35 Melanoma cell lines hybridized to Affymetrix Hu133_Plus 2 microarrays were analysed for genes differentially expressed between cell lines carrying wild-type p14ARF and those with mutant 14ARF. All of these cell lines contained wild-type p53 (so that the effects of p14ARF mutations could be analysed without contamination from p53). Experiment Overall Design: The aim of this study was to identify downstream effectors of p14ARF in melanoma. Relevant paper: Packer et al. (2007) Int J Cancer.
Project description:Exosomes are small membraneous vesicles secreted into body fluids by tumors. Tumor exosomes contain intact and functional mRNAs, small RNAs (including miRNAs), and proteins that can alter the cellular environment to favor tumor growth. Further exploration into the molecular profiling of exosomes may increase our understanding of their roles in melanoma progression in vivo, and may have potential application in biomarker studies. In the present study, we used mRNA array profiling to identify thousands of exosomal mRNAs associated with melanoma progression and metastasis. Similarly, miRNA array profiling identified specific miRNAs, such as hsa-miR-31, -185, and -34b, involved in melanoma invasion. Our results indicate that melanoma-derived exosomes have unique gene expression signatures and miRNA profiles that may have important functions in melanoma metastasis and progression. Total RNA from cells and exosomes were isolated using mirVana total RNA isolation kit according to the manufacturer’s guidelines. RNA was quantified using Nanodrop ND-1000. The integrity of these total RNAs was assessed using Agilent 2100 Bioanalyzer. Total high-quality RNA was converted to cDNA, transcribed and labelled, and then hybridized to human HG-U133 plus 2 arrays (Affymetrix) then scanned according to the standard protocol recommended by Affymetrix. Two different RNA preparations from two cell lines and their exosomes were used.
Project description:Melanomas are often infiltrated by activated inflammatory cells. Thus, melanoma cells are very likely stimulated by inflammatory cytokines. In order to assess the impact of common inflammatory cytokines, we investigated the gene expression profile of melanoma cell lines before and after cytokine treatment in vitro. Experiment Overall Design: 5 human melanoma cell lines were treated with either IFN-α 1,000 U/ml, IFN-γ 100 U/ml or TNF-α 10 ng/ml for 72 hours, or were left untreated. We analyzed their expression profile with Affymetrix expression arrays.