Noncoding somatic and inherited single-nucleotide variants converge to promote ESR1 expression in breast cancer
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Mathieu Lupien
PROVIDER: E-GEOD-74718 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Next-generation sequencing has revolutionized cancer biology by accelerating the unbiased discovery of novel mutations across human cancers. Transforming such discoveries into a conceptual framework of cancer progression requires narrowing the vast number of mutations down to the driver elements, and further reducing these to mutations that govern cancer progression as opposed to tumor initiation. By integrating next-generation RNA-sequencing (RNA-seq) with in vivo selection, we devise an approach that identifies a series of novel recurrent non-synonymous amino acid mutations that are enriched in metastatic breast cancer cells and predicted to significantly alter protein function. These mutations, found in PANX1, RBFA, REST, KRIT1 and ZSWIM6, are detected at higher frequencies in the transcriptomes of two patientsâ highly metastatic sub-lines relative to their poorly metastatic parental lines. We functionally characterize the cellular and molecular roles of one of these mutationsâa nonsense alteration that yields a truncated pannexin-1 (PANX11-89) plasma membrane megachannel subunitâin metastatic progression. PANX11-89 interacts with full-length PANX1 and augments PANX1 channel activity to promote the survival of cancer cells as they are mechanically deformed. Protection from deformation-induced cell death requires PANX1 channels to release ATP, which acts as a cell autonomous survival signal during mechanical stress. Functional characterization of additional nonsense and missense PANX1 mutations detected in epithelial cancers of the colon, lung, and prostate reveals that these mutants also enhance PANX1-mediated ATP release. In vivo testing of one such truncating mutation detected in a metastatic colorectal tumor also enhances early survival, dissemination and liver metastatic colonization by human colon cancer cells. Finally, pharmacological inhibition of PANX1 inhibits breast cancer metastasis, implicating PANX1 as a novel therapeutic target in cancer. Our findings reveal that ATP release through mechanosensitive PANX1 channels enables cancer cells to overcome a major metastasis suppressive barrier—deformation-induced death in the microvasculature. To systematically identify base-pair mutations present in metastatic cells that may drive cancer progression, we performed whole-transcriptome RNA-sequencing (RNA-seq) of in vivo-selected, highly metastatic human breast cancer cell sub-lines, CN-LM1A and MDA-LM2, as well as the CN34 and MDA-MB-231 parental lines from which they were derived. To minimize the false positive rate and allow for subsequent statistical analysis, we sequenced biological replicates of each cell line. Mutations conferring enhanced metastatic capacity should be enriched in the transcriptomes of highly metastatic cells relative to their less metastatic parental populations.
2015-06-22 | E-GEOD-45162 | biostudies-arrayexpress
Project description:To study mixotrophy, it is desirable to have an organism capable of growth in the presence and absence of both organic and inorganic carbon sources, as well as organic and inorganic energy sources. Metallosphaera sedula is an extremely thermoacidophilic archaeon which has been shown to grow in the presence of inorganic carbon and energy source supplements (autotrophy), organic carbon and energy source supplements (heterotrophy), and in the presence of organic carbon and inorganic energy source supplements. The recent elucidation of M. sedulaâ??s inorganic carbon fixation cycle and its genome sequence further facilitate its use in mixotrophic studies. In this study, we grow M. sedula heterotrophically in the presence of organic carbon and energy sources (0.1% tryptone), autotrophically in the presence of inorganic carbon and energy sources (H2 + CO2), and â??mixotrophicallyâ?? in the presence of both organic and inorganic carbon and energy sources (0.1% tryptone + H2 + CO2 ) to characterize the nature of mixotrophy exhibited. Two 3 slide loops joined at equivalent conditions (8 slides total) for Mse cells includes 3 conditions tested in duplicate (biological repeats): heterotrophy (H1 and H2), autotrophy (A1 and A2), and mixotrophy (M1 and M2). Half of an RNA sample for one condition was labeled with Cy3 while the other half was labeled with Cy5. The two differently labeled samples were run on different slides. Each probe is spotted on each slide 5 times (5 replicates; spot intensities for all replicates on slide provided in associated raw data file).
2010-06-20 | E-GEOD-14978 | biostudies-arrayexpress
Project description:Using criteria based on known examples, we identified approximately 1.7 million and 2.4 million sequences encoding putative miRNA precursor (stem-loop) structures in the mouse and human genomes, respectively, as well as large numbers of such structures in the genomes of fish, fruitfly and nematode worm. These predictions were then tested using high density custom microarrays containing modified oligonucleotides interrogated with purified small RNAs from different tissues. We found that 19% of 4,006 randomly chosen mouse predictions and 40% of 1,859 randomly chosen human predictions gave positive signals with small RNA isolated from whole 10-12 day embryos and a mixture of brain and testis, respectively, 84% of which hybridized to only one strand of the predicted stem-loop structure, whereas only 2% and 12% of equivalent random mouse and human predictions were positive. There was no difference in the validation rates between sequences exhibiting conservation and those that did not. High validation rates were also observed with predictions from fish, fly and worm genomes. Northern blot analysis of the array-positive mouse predictions confirmed that the vast majority of these sequences were detectable as small RNAs whose sizes ranged from 20-110 nucleotides. Taking into account false negative rates of the prediction and detection of known miRNAs, and assuming that this holds for other small RNAs, we estimate that there are 1 million small RNAs expressed in mouse and 3 million small RNAs in human. Keywords: genomic survey of small RNAs Stemloops were identified in the mouse and human genome using RNALfold with a window size of 110nt. Folding energy and other criteria were used to filter the structures into a set of miRNA-like stems. Stems were chosen randomly and probes designed against the stem regions. Stems were called present if one of the stem probes was more than 4 standard deviations above the signal distribution of mRNA degradation contamination probes designed against GAPDH and beta-actin.
2010-05-18 | E-GEOD-7217 | biostudies-arrayexpress
Project description:Developmental exposure of mouse fetuses to estrogens results in dose-dependent permanent effects on prostate morphology and function. Fetal prostatic mesenchyme cells express estrogen receptor alpha (ERα) and androgen receptors and convert stimuli from estrogens and androgens into signaling to regulate epithelial cell proliferation and differentiation. To obtain mechanistic insight into the role of different doses of estradiol (E2) in regulating mesenchymal cells, we examined E2-induced transcriptomal changes in primary cultures of fetal mouse prostate mesenchymal cells. Urogenital sinus mesenchyme cells were obtained from male mouse fetuses at gestation day 17 and exposed to 10 pM, 100 pM or 100 nM E2 in the presence of a physiological concentration of dihydrotestosterone (0.69 nM) for four days. Gene ontology studies suggested that low doses of E2 (10 pM and 100 pM) induce genes involved in cell adhesion, morphological tissue development, and sterol biosynthesis but suppress genes involved in growth factor signaling and cell adhesion. Genes showing inverted-U-shape dose responses (enhanced by E2 at 10 pM E2 but suppressed at 100 pM) were identified, and their enrichment in the glycolytic pathway was demonstrated. At the highest dose (100 nM), E2 induced genes enriched not only for cell adhesion but also steroid hormone signaling and metabolism, cytokines and their receptors, cell-to-cell communication, Wnt signaling, and TGF-β signaling. These results suggest that prostate mesenchymal cells may regulate epithelial cells through direct cell contacts when estrogen level is low whereas soluble growth factors might play significant roles when estrogen level is high. Primary culture urogenital sinus mesenchymal cells were isolated from prostate glands of gestation day 17 CD1 male mouse fetuses. Cells were then exposed to 10 pM or 100 pM 17beta-estradiol or vehicle (0.05% ethanol) for four days in the presence of 690 pM 5alpha-dihydrotestosterone. Total cellular RNA was then isolated for determination of transcriptomal profiles by Affymetrix mouse gene 1.0 ST array.
2012-03-21 | E-GEOD-36630 | biostudies-arrayexpress
Project description:Background Clinical success of T cell receptor (TCR) gene therapy has previously been demonstrated for NY-ESO-1 TCR gene therapy. To increase numbers of cancer patients that can be treated with TCR gene therapy we aimed to identify a novel set of high-affinity cancer specific TCRs targeting different cancer testis (CT) antigens in prevalent HLA class I alleles. Methods In this study, we selected based on publicly available gene expression databases the most promising CT genes to target. From these selected genes we identified by HLA peptidomics the naturally processed and presented HLA class I peptides. With these peptide-HLA tetramers were generated, and by single cell sorting CT specific CD8+ T cells were selected, and expanded from the allo-HLA repertoire of healthy donors. By several functional assays high avidity CT-specific T cell clones with safe recognition pattern were selected. To evaluate the potential for clinical application in TCR gene therapy, TCRs were sequenced and transferred into peripheral blood derived CD8+ T cells. Results In total we identified, 7 novel CT-specific TCRs that effectively target MAGE-A1, MAGE-A3, MAGE-A6 and MAGE-A9 in the context of human leukocyte antigen(HLA) -A1, -A2, -A3, -B7, -B35 and -C7. TCR gene transfer into CD8⁺ T cells resulted in efficient cytokine production and cytotoxicity of variety of different tumor types without detectable cross-reactivity. In addition, major in vivo antitumor effects of MAGE-A1 specific TCR engineered CD8⁺ T cells was observed in an orthotopic xenograft model for established multiple myeloma, in which bone marrow located tumor cells were completely eradicated after T cell injection. Conclusion The identification of 7 novel CT-specific TCRs, reactive against CT antigens presented in a variety of different HLA class I alleles, allows selection of therapeutic TCRs for an increased number of cancer patients, and will improve development of personalized TCR gene therapy.
2023-01-23 | PXD038862 | Pride
Project description:In order to establish a list of candidate direct COUP-TFI gene targets in the inner ear, we analyzed the differential gene expression profiles of the wild-type and the COUP-TFIâ/â P0 inner ears. We performed a total of 8 microarray experiments using Affymetrix MG-U74Av2 chips, including 2 biological and 2 experimental replicates per genotype sample. Due to limiting RNA yields from the newborn inner ear, each microarray chip was hybridized with an RNA pool from multiple tissue samples. We analyzed our data using two different algorithms: GC-Robust Multi-Array (GCRMA) and dChip. Each normalized expression dataset was subsequently analyzed by 2-way ANOVA, evaluating both genotype and experimental effects. This statistical approach allowed us to 1) account for an experimental effect observed in the expression value of many genes, therefore increasing the power of the analysis, and 2) filter out potential expression differences due to contamination during dissections (contaminating genes would present as probes with a significant interaction p-value). Using this methodology, the gene hits from the GCRMA-normalized expression dataset consisted of 256 genes with a significant genotype effect (p<0.01) and no interaction (p>0.01). Similar cutoffs applied on the dChip-normalized dataset resulted in 250 significant gene hits. Within both groups, COUP-TFI has the lowest genotype p-value, validating our statistical approach.
2010-06-08 | E-GEOD-16744 | biostudies-arrayexpress
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2012-09-02 | E-GEOD-35406 | biostudies-arrayexpress
Project description:Investigation of baseline transcription activity of two different clinical isolates of Staphylococcus aureus with two different susceptibility levels to the antibiotics Vancomycin and Daptomycin. Two different strains of Staphylococcus aureus, one that is fully Vancomycin and Daptomycin Sensitive and one with decreased Vancomycin and Daptomycin Sensitivity - grown to mid-log phase in rich broth.
2012-08-03 | E-GEOD-39868 | biostudies-arrayexpress
Project description:Histone modifications are important markers of function and chromatin state, yet the DNA elements that direct them to specific locations in the genome are poorly understood. Here we use the genetic variation in Yoruba lymphoblastoid cell lines as a natural experiment to identify genetic differences that affect histone marks and to better understand their relationship with transcriptional regulation. Across the genome, we identified hundreds of quantitative trait loci that impact histone modification or RNA polymerase (PolII) occupancy. In many cases the same variant is associated with quantitative changes in multiple histone marks and PolII, as well as in DNaseI sensitivity and nucleosome positioning, indicating that these molecular phenotypes often share a single underlying genetic cause. Variants that impact chromatin at distal regulatory sites frequently also direct changes in chromatin and gene expression at associated promoters; while most of these distal regulators enhance promoter activity, some act as distal chromatin silencers. Finally, we find that polymorphisms in transcription factor binding sites are often causally responsible for variation in local histone modification. In summary, the class of variants identified here generate coordinated changes in chromatin both locally and sometimes at distant locations, frequently drive changes in gene expression, and likely play an important role in the genetics of complex traits. ChIP-seq of RNA Polymerase II and 4 histone modifications (H3K4me1, H3K4me3, H3K27ac, H3K27me3) in 10 unrelated Yoruba HapMap lymphoblastoid cell lines
2013-10-01 | E-GEOD-47991 | biostudies-arrayexpress
Project description:Cyclin-dependent kinases 4 and 6 (CDK4/6) are essential drivers of the cell cycle and are also critical for the initiation and progression of diverse malignancies. Pharmacological inhibitors targeting CDK4/6 have demonstrated significant activity against various tumor types such as breast cancer. However, resistance to CDK4/6 inhibitors (CDK4/6i) (such as palbociclib) remains an immense obstacle in clinical and the underlying mechanisms have not been fully understood. Using quantitative high-throughput combinational screen (qHTCS) and genomic sequencing, we report that the Microphthalmia-associated transcription factor (MITF), was significantly elevated in palbociclib-resistance cells. Inhibition of MITF can enhance the therapeutic efficacy of Palbociclib and surmount Palbociclib resistance both in vitro and in vivo. Mechanistically, we found that O-GlcNAc transferase (OGT) modifies MITF with O-GlcNAcylation at Serine 49 (Ser49) within its nuclear localization signal (NLS), thereby promoting MITF binding to importin α/ β and facilitating its nuclear transportation, which is crucial in regulating senescence.
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