Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Module map of stem cell genes guides creation of epithelial cancer stem cells

ABSTRACT: Self-renewal is a hallmark of stem cells and cancer, but existence of a shared stemness program remains controversial. Here we construct a gene module map to systematically relate transcriptional programs in embryonic stem cells (ESCs), adult tissue stem cells, and human cancers. This map reveals two predominant gene modules that distinguish ESCs and adult tissue stem cells. The ESC-like transcriptional program is activated in diverse human epithelial cancers and strongly predicts metastasis and death. c-Myc, but not other oncogenes, is sufficient to reactivate the ESC-like program in normal and cancer cells. In primary human keratinocytes transformed by Ras and IkBa, c-Myc increases the fraction of tumor-initiating cells by 150-fold, enabling tumor formation and serial propagation with as few as 500 cells. c-Myc-enhanced tumor initiation is cell-autonomous and independent of genomic instability. Thus, activation of an ESC-like transcriptional program in differentiated adult cells may induce pathologic self-renewal characteristic of cancer stem cells. A genetic modification design type is where an organism(s) has had genetic material removed, rearranged, mutagenized or added, such as knock out. Computed

ORGANISM(S): Homo sapiens

SUBMITTER: David Wong

PROVIDER: E-GEOD-10423 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Module map of stem cell genes guides creation of epithelial cancer stem cells.

Wong David J DJ Liu Helen H Ridky Todd W TW Cassarino David D Segal Eran E Chang Howard Y HY

Cell stem cell 20080401 4

Self-renewal is a hallmark of stem cells and cancer, but existence of a shared stemness program remains controversial. Here, we construct a gene module map to systematically relate transcriptional programs in embryonic stem cells (ESCs), adult tissue stem cells, and human cancers. This map reveals two predominant gene modules that distinguish ESCs and adult tissue stem cells. The ESC-like transcriptional program is activated in diverse human epithelial cancers and strongly predicts metastasis an ...[more]

PMID: 18397753

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Project description:To identify mRNAs associated with human PUM2 protein, we used a modified Ribonucleoprotein-ImmunoPrecipitation Microarray (RIP-Chip) approach on HeLa S3 cancer cells that express PUM2. PUM ribonucleoprotein (RNP) complexes were captured from cell-free extracts with specific anti PUM2 antibody coupled to protein A sepharose beads, and then eluted with SDS-EDTA. To control for non-specifically enriched RNAs, the same procedure was performed with beads that were not coupled with immunoprecipitating antibodies (mock samples). Total RNA was isolated from cell extracts and immunopurified samples with the mirVanaTM PARISTM kit (Ambion). RNA was quantified with a NanoDrop device (Witeg AG). Poly-adenylated RNAs were amplified in the presence of aminoallyl-UTP with Amino Allyl MessageAmp II aRNA kit (Ambion). For this purpose, 500 ng total RNA from extracts and half (50-100 ng) of the immunopurified RNAs were used for amplification. 8 ug of the amplified RNAs (aaRNA) were fluorescently labeled with NHS-monoester Cy3 and Cy5 dyes (GE HealthSciences), except for mock RNA samples, where an aaRNA amount proportional to the yield obtained from corresponding PUM affinity isolates was used.For PUM2 RIPs, we performed four biological replicates but omitted the dye swaps due to the lower aaRNA obtained after amplification (~10 ug aaRNA from PUM2 RIPs, 9 ug aaRNA from mock RIPs). The Cy3- and Cy5-labeled aaRNA samples were mixed and hybridized to human cDNA microarrays. Detailed methods for microarray experiments are available at http://cmgm.stanford.edu/pbrown/protocols/index.html. cDNA microarrays were produced by the Stanford Functional Genomic Facility and contained 43,197 human probes representing 26,524 Unigene cluster IDs (12,466 ENSEMBL annotated genes) spotted on Corning Ultra GAPS slides. Spotted cDNAs were cross-linked with 65 mJ of UV irradiation on slides, which were then post-processed for 1 hour at 42°C in pre-hybridization solution (5x SSC, 0.1% SDS, 0.1 mg/ml BSA), washed twice in 400 ml of 0.1x SSC for 5 min, dunked in 400 ml ultrapure water for 30 sec, and dried by centrifugation at 550 rpm for 5 min. Slides were used the same day. Cy3- and Cy5-labeled aaRNA probes were mixed and applied to arrays in hybridization solution (3x SSC, 20 ug poly(A) RNA [Invitrogen], 20 ug yeast tRNA [Invitrogen], 20 ug Human Cot-1 DNA [Invitrogen], 20 mM HEPES [pH 7.0] and 0.3% SDS) for 18 h at 65°C. The arrays were then washed sequentially in 400 ml of 2x SSC with 0.1% SDS, 1x SSC, and 0.2x SSC. The first wash was performed for 5 min at 65°C, the subsequent washes were performed for 5 min at RT. The arrays were dried by centrifugation and immediately scanned with an AxonScanner 4200A (Molecular Devices). Data were collected using GENEPIX 5.1 (Molecular Devices). Arrays were normalized computationally by the Stanford Microarray Database (SMD). The data were filtered for signal over background of greater than 1.5 in the channel measuring aaRNA from extract, and only features that met these criteria in > 50% of the arrays were included for further analysis. Log2 median ratios were retrieved and exported into Microsoft Excel. To identify transcripts that were specifically enriched by association with PUM2, we performed two class Significance Analysis of Microarrays (SAM) on median centered arrays. A replicate experimental design type is where a series of replicates are performed to evaluate reproducibility or as a pilot study to determine the appropriate number of replicates for a subsequent experiments. Antibody used in IP: 50 ug rabbit anti-PUMILIO 2 (Bethyl Laboratories, #300-202A) Keywords: replicate_design Computed

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Module map of stem cell genes guides creation of epithelial cancer stem cells

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Module map of stem cell genes guides creation of epithelial cancer stem cells.

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