Deciphering targets of CD271-dependent signaling processes
ABSTRACT: Genome-wide expression profiling of stably NGFR transfected melanoma cells was used to identify genes driven by expression of the nerve growth factor receptor CD271 (NGFR). Stable overexpression of NGFR (CD271): Generation of cell lines stably overexpressing CD271 (NGFR), melanoma cells were transfected with 2 µg of a plasmid expressing GFP-tagged human NGFR (RG207966, OriGene) and selected with G418 (100-300 µg/ml, PAA) over a period of two weeks followed by sub-cloning or FACS. Gene expression profiling: Whole genome expression profiling of T20/02 and A375 cells (NGFR) and control cells (Mock or GFP) was performed with three biological replicates. Illumina raw data of BeadChip HumanHT-12V4 platform were summarized via the BeadStudio without normalization and background correction. Follow-up processing was done via the R/Bioconductor environment employing packages lumi, limma and q-value. Data were normalized with quantile normalization. Genes were termed significantly differentially expressed when the average detection p-value of at least one case was < 0.05 the ratio was outside the interval [0.75,1.33], one of the p-values from limma test, Student's t-test, Welch test and Wilcoxon test was < 0.05. At least one of the q-values corresponding to one of these tests < 0.05.
Project description:Background: Large-scale genomic analyses of patient cohorts have revealed extensive heterogeneity between individual tumors, contributing to treatment failure and drug resistance. In malignant melanoma, heterogeneity is thought to arise as a consequence of the differentiation of melanoma-initiating cells that are defined by cell-surface markers like CD271 or CD133. Results: Here we identified the nerve growth factor receptor (CD271) as a crucial determinant of melanoma cell tumorigenicity, stem-like properties, heterogeneity and plasticity. Stable shRNA mediated knock-down of CD271 in patient-derived melanoma cells abrogated their tumor-initiating and colony-forming capacity. A genome-wide expression profiling and gene-set enrichment analysis revealed novel connections of CD271 with melanoma-associated genes like CD133 and points to a neural crest stem cell (NCSC) signature lost upon CD271 knock-down. In a meta-analysis, we found CD271 linked to the neural crest specifier SOX10 and observed a shared set of 271 differentially regulated genes. To dissect the connection of CD271 and CD133 we analyzed 10 patient-derived melanoma-cell lines for cell-surface expression of both markers compared to established cell lines MeWo and A375. We found CD271+ cells in the majority of cell lines analyzed as well as in a set of 16 different patient-derived melanoma metastases. Strikingly, only 2/12 cell lines harbored a CD133+ sub-set that in addition comprised a fraction of cells of a CD271+/CD133+ phenotype. Those cells were found in the label-retaining fraction and in vitro deduced from CD271+ but not CD271 knock-down cells. Conclusions: Our present study provides a deeper insight into the regulation of melanoma cell properties and points CD271 out as a regulator of several melanoma-associated genes. Further, our data strongly suggest CD271 is a crucial determinant of stem-like properties of melanoma cells like colony-formation and tumorigenicity. For knock-down of CD271 (NGFR), melanoma cells were transfected with 2 µg of each shRNA plasmid; #2: 5’-ACAACCTCATCCCTGTCTATT-3’; #3: 5’-CCCGAGCACATAGA CTCCTTT-3’ and #4: 5’-CCGAGCACATAGACTCCTTTA-3’ or control shRNA (shCtl 5’-GGAATCTCATTCGATGCATAC-3’; all from Quiagen) using Lipofectamine2000 (Invitrogen). Cells were selected with puromycin (10µg/ ml) over a period of two weeks. Whole genome expression profiling of CD271k.d. cells (shRNA#4) and shCtl. cells was performed with three biological replicates. Illumina raw data of BeadChip HumanHT-12V4 platform were summarized via the BeadStudio without normalization and background correction. Follow-up processing was done via the R/Bioconductor environment employing packages lumi, limma and q-value. Data were normalized with quantile normalization. Genes were termed significantly differentially expressed when the average detection p-value of at least one case was < 0.05 the ratio was outside the interval [0.75,1.33], one of the p-values from limma test, Student's t-test, Welch test and Wilcoxon test was < 0.05. At least one of the q-values corresponding to one of these tests < 0.05.
Project description:Some cancers are thought to follow a cancer stem cell (CSC) model in which hierarchical and epigenetically determined relationships exist between phenotypically and functionally distinct cells within tumors. In melanoma, for example, CD271/p75/NGFR (nerve growth factor receptor) was found by some groups to be expressed in cells with enriched tumorigenic potential (Boiko et al., 2010; Civenni et al., 2011). However, these observations were not reproduced in highly efficient patient-derived xenograft (PDX) assays (Quintana et al., 2008), in which plastic, nonhierarchical relationships were proposed between CD271- and CD271+ cells (Quintana et al., 2010). Here we confirm that a CD271-based CSC model does not apply to melanoma, regardless of the PDX assay method used. In side-by-side testing of published PDX assays, no consistent differences were seen in CD271 expression or in PDX tumor formation from purified CD271- or CD271+ cells from patient melanomas. Analysis of CD271 in sixty-eight PDX tumors grown from CD271- or CD271+ cells revealed strikingly variable CD271 re-expression patterns, which were not consistent with stable hierarchical or plastic relationships between CD271-defined cell sub-populations. Genotyping of sibling PDX tumors showed extensive (28% - 48%) genomic copy number differences, which were also apparent (1.4% - 23%) between CD271- and CD271+ cells purified from each of four melanomas, revealing intra-tumoral genetic heterogeneity associated with CD271 expression. Thus, CD271 is not linked reproducibly to tumorigenic potential in melanoma, regardless of the method of melanoma cell isolation, and its expression appears driven by complex and unstable interactions between changing epigenetic and genetic states. Understanding of melanoma biology and the treatment of melanoma patients are unlikely to be advanced by applying a CD271-based CSC model to this disease. Overall design: To test whether differences in CD271 expression among PDX melanomas grown from CD271- or CD271+ cells could be linked to genetic differences, we genotyped Lin- cells sorted by flow cytometry from four pairs of tumors. Tumors in each pair were grown from aliquots from the same pools of cells, purified according to CD271 expression from patient melanomas 12-1036 and 12-1254. Genomic DNA was extracted from purified Lin- cells from each tumor and subjected to single nucleotide polymorphisms (SNP) genotyping using Illumina Human OmniExpress 715K arrays. To test for genetic differences between CD271- and CD271+ cells within the same tumor, we flow cytometrically-purified CD271- and CD271+ cells from patient (n=1) and PDX (n=4) melanomas, extracted DNA and subjected to SNP genotyping using Illumina 2.58M arrays.
Project description:Human engeneered skin carrying GFP positive melanoma cells was transplanted in immunocompromised rats. 6 weeks after transplatation the engeenered skin was removed and GFP positive melanoma cells sorted for CD271 HIGH and LOW populations Overall design: 2 rats were transplanted, the 2 tranplants were then dissected and cell isolated by digestion. Melanoma cells were sorted for GFP Functional Genomic Center University of Zürich Switzerland for Affymetrix and data analysis
Project description:EWI-2 (IgSF8) plays a novel, bifunctional role in melanoma cells. EWI-2 inhibits migration, metastasis, EMT-like changes, and CD271-dependent invasion in multiple melanoma cell lines. On the other hand, EWI-2 supports melanoma cell proliferation, survival, and xenograft growth. Consistent with these results, EWI-2 levels were elevated in human malignant melanoma, but not in metastatic melanoma samples. Altered melanoma cell functions, caused by EWI-2 ablation, are almost entirely dependent on enhanced TRF-β1 signaling, and also require contributions from tetraspanin proteins CD9 and CD81. In melanoma cells lacking EWI-2, tetraspanins CD9 and CD81 enhance TRF-β1 signaling by facilitating TβR-2−TβR-1 receptor complex formation. When EWI-2 is present, CD9 and CD81 are diverted into EWI-2 complexes, and thus TRF-β1 signaling is inhibited. 4 samples = 2 Control + 2 EWI-2 KD.
Project description:We report the gene expression profiles by NGFR knockdown in H460 and H1299 cell lines and reveal that NGFR ablation activates p53 target gene expression. We examined gene expression in two different non-small-cell lung cancer cell lines, one with wild-type p53 and the other without p53.
Project description:We used transcription-profiling to identify mitogen-activated protein kinase (Mapk) signaling as an important regulator involved in the differentiation of oligodendrocyte progenitor cells (OPCs) into oligodendrocytes. We show in tissue culture that activation of Mapk signaling by elevation of intracellular levels of cAMP using administration of either dibutyryl-cAMP or inhibitors of the cAMP-hydrolyzing enzyme phosphodiesterase-4 (Pde4) enhances OPC differentiation. Finally, we demonstrate that systemic delivery of a Pde4 inhibitor leads to enhanced differentiation of OPCs within focal areas of toxin-induced demyelination and a consequent acceleration of remyelination. Study was conducted to identify the genes that are differentialy regulated during early time point of OPC differentiation. This study includes reanalyzed data from the following 6 Samples available in GSE24821: [i] 5 days post lesion (dpl) laser captured caudal cerebellar peduncle (CCP) replicates 1-3 (GSM610956-GSM610958), and [ii] 14 dpl laser captured CCP replicates 1-3 (GSM610959-GSM610961). For the reanalyzed data, raw data files were background-corrected, normalized and summarized using the RMA algorithm as implemented in the R statistical programming environment (http://www.r-project.org). Differential expression between sample groups was assessed using the moderated t-test as implemented in the limma package for the Bioconductor suite of R bioinformatics software (http://bioconductor.org). In order to correct for hypothesis testing on such a scale, P-values relating to differential expression were corrected for multiple testing using FDR correction. Differential expression across sample groups was deemed significant at an FDR of 5% (q < 0.05). The processed data for the reanalyzed dataset is linked below as supplementary file 'GSE50042_reanalyzed_Samples.txt'. The processed data for Samples GSM1213100-GSM1213114 is linked below as supplementary file 'GSE50042_processed_data.txt'.
Project description:In order to gain a better understanding of the effects of Streptomyces coelicolor A3(2) wild type strain exposed to simulated microgravity condition by clinorotation, we constructed a whole genome microarray to screen simulated microgravity sensitive genes using the Agilent eArray 5.0 program. The genechip was custom designed according to the manufacturer’s recommendations (Agilent, 8*15K). The genome sequences were downloaded from: http://www.ncbi.nlm.nih.gov/genome?Db =genome&Cmd = Search&Term = NC_003888, NC_003903.1, NC_003904.1. Each gene was represented by one 60-nt oligonucleotide probe, and 680 genes associated with morphological differentiation and secondary metabolism out of the total 8116 genes were replicated 9 times each. Cells of Streptomyces coelicolor A3(2) wild type strain were harvested from the colonies grown in JCM42 agar medium at 28°C for 96h (4d) under simulated microgravity and 1g control conditions. Total RNA was extracted to conduct the microarray hybridization experiments. Three independent experiments were performed in which ASM_1A, ASM_2A, ASM_3A were the test group samples and ANG_1A, ANG_2A, ANG_3A were the control group samples. Differentially expressed genes were selected with P＜0.05, FC≥2.0 by T-test methods using the independent three sets data.
Project description:Surgical glaucoma therapy is characterized by implantation of an aqueous shunt either draining into the extraocular Tenon’s space or the intraocular suprachoroidal space. In both cases the long term drainage is hampered by fibrotic reactions around the outflow region of the shunt. The prevention of fibrosis should extend the operating life of the shunt. For an aqueous shunt draining from the anterior chamber into the choroidal space fibroblasts from the choroidea and/or the sclera are most likely responsible for a fibrotic response around the outflow region of such a shunt. A detailed characterization of fibroblasts derived from choroidea and sclera should provide information whether a fibrosis reaction can be inhibited by cell type specific agents. Therefore, we have decided to generate mRNA profiles of fibroblasts from the choroidea, sclera and Tenon’s space in order to look for potential pharmacological targets for fibrosis prevention. The three fibroblast types investigated share fibroblast specific gene expression patterns, concerning extracellular matrix proteins as collagens and fibronectin, but also show distinct mRNA patterns, which we plan to search for targets responsible for fibrotic processes which hopefully can be targeted by specific antifibrotic drugs. Three human fibroblast cell type cultures from different ocular tissues were established: sclera fibroblasts (hSF), choroidea fibroblasts (hCF), and Tenon’s space fibroblasts (hTF). For the gene expression analysis n = 5 for hCF, n = 4 for hSF, and n = 5 for hTF donor cells were cultivated from different donors. After appropriate cultivation, cells were harvested, RNA was extracted, purified and quantity and quality was assessed. All total RNA samples were analyzed by Affymetrix' Whole-Transcript Expression Analysis & Profiling Human Gene ST Arrays, respectively. In this set-up, we run = 5 arrays for hCF, n = 4 arrays for hSF, and n = 5 arrays for hTF i.e. one array per biological replicate. No technical replication was carried out. Microarray data analysis was carried out by using the Rosetta Resolver® system for gene expression data analysis (Rosetta Biosoftware, Seattle, WA, USA). In brief, the raw signals of the probes were summarized using RMA thereby generating probe set specific signal intensities. Chips were normalized by using quantile normalization. To compare RNA expression levels of genes in hCF, hSF and hTF, normalized expression signals of genes from corresponding samples were averaged and fold changes were calculated. To assess differences in mean signal intensities between experimental groups, ANOVA (analysis of variance, with Benjamini Hochberg test correction) and a post-hoc Scheffe test was performed. Rosetta Resolver ratio built statistics to correct for possible signal intensity bias were also considered. Only genes (1) an absolute fold change of ≥ 1.5 together with a Scheffe test p value ≤ 0.05 in at least one of the three pairwise comparisons hCF vs. hTF, hSF vs. hTF and hCF vs. hSF, resp., as well as (2) a ratio built p value ≤ 0.05 were deemed differentially expressed genes (DEG) and considered for further evaluation.
Project description:Analyze and compare the gene expression profile of human bone marrow primary CD45-CD271+ and CD45-CD271- cells. The hypothesis was some genes were differentially expressed in these two populations. Results provided important information regarding the gene expression difference of these two cell populations. Overall design: Total RNA obtained from isolated CD45-CD271+ and CD45-CD271- cells were amplified and genome wide gene profiling was compared.
Project description:Purpose: We have succeeded in the generation and long-term expansion of SOX9-expressing CD271+PDGFRa+CD73+ chondrogenic ectomesenchymal cells from the PAX3/SOX10/FOXD3-expressing MIXL1-CD271hiPDGFRaloCD73- neural crest-like progeny of human pluripotent stem cells in a chemically defined medium supplemented with Nodal/Activin/TGFb inhibitor (SB) and FGF2 (hereafter called FSB). When “primed” with TGFb, such cells efficiently formed translucent cartilage particles, which were completely mineralized in 12 weeks in immunocompromized mice. Under the FSB condition, ectomesenchymal cells were expandable without loss of chondrogenic potential for at least 16 passages, maintained normal karyotype for at least 10 passages, which any conditions deviated from it (e.g. FGF2 alone or SB alone) failed to support. In order to address the molecular basis of such effects of FSB, a series of RNA-seq experiments were carried out. Methods: We generated and compared the transcriptome profiles of human ectomesenchymal cells expanded under FSB with those cultured under FSB first then under FGF2 alone (F). As a control, we also generated transcriptome of ectomesenchymal cells expanded from the begining under F conditions. RNA-sequencing libraries were prepared using a SureSelect Strand Specific RNA Library Preparation kit (Agilent technologies, Santa Clara, CA). Sequencing was performed on an Illumina HiSeq 1500 using a TruSeq Rapid SBS kit (Illumina, San Diego, CA) in a 50-base single-end mode. Sequenced reads were mapped against the human reference genome (GRCh37), using TopHat v2.0.12 (http://ccb.jhu.edu/software/tophat/index.shtml). Expression levels were calculated as fragments per kilobase of exon per million mapped fragments (FPKMs) using Cufflinks v2.1.1 (http://cole-trapnell-lab.github.io/cufflinks). Results: Ectomesenchymal cells maintained under FSB conditions preferentially expressed genes representing embryonic progenitors (SOX4/12, LIN28A/B, MYCN), cranial mesenchymes (ALX1/3/4) and chondroprogenitors (SOX9, COL2a1) of the neural crest origin (SOX8/9, NGFR, NES). In contrast, those cultured under FSB then F, still expressed SOX4/11/12, but lost LIN28, MYCN, ALX1/3/4, NGFR, COL2a1 expression. Interestingl it enhances expresion ofTGFβ-inducible genes such as THBS1/2 and DCN and osteogenesis-related genes such as COL1a1/2 and RUNX1/2. Conclusions: The CD271+CD73+ ectomesenchymal cells accumulated under FSB conditions possess an mRNA profile of proliferating primitive neural crest/ectomesenchymal cells, although they lacked SOX10 expression, which is critical for neural and melanocytic lineage commitment. Transition from FSB to F conditions supressed the proliferation-related genes expression and enhanced the ossification/mineralization, vasculogenesis/angiogenesis, and cardiac myogenesis-related gene expression. Thus, suppression of TGFβ signaling by SB does not seem to freeze the developmental stage of the hPSC-derived neural crest during expansion. Such suppression may instead simply support the high proliferative potential of the cells as well as the expression of SOX9 (and COL2a1), and thereby maintain chondrogenic activity. SOX9 expression initiated at the specification and pre-migratory stages is transient in trunk neural crest but persists in cranial neural crest. The chondrogenic CD271+CD73+ ectomesenchymal cells that maintain SOX9 transcription and translation may therefore represent proliferating cranial neural crest, with a slight commitment to non-neural lineages. Examination of human ES-derived neural crest-like progenies expanded in 3 different culture media. Each group contains three biological replicates.