Ena-DATASET-MUG-22-01-2021-21:15:57:448-180 - samples
Ontology highlight
ABSTRACT:
PROVIDER: EGAD00001006897 | EGA |
REPOSITORIES: EGA
Similar Datasets
Project description:Mutation analysis in plasma samples with low ctDNA levels using a molecular barcoding technology, i.e. the single target approach SiMSen-seq (Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing).
| EGAD00001006104 | EGA
Project description:Oncogenic PIK3CA mutations activate phosphoinositide 3-kinase (PI3K) and are among the commonest somatic mutations in cancer and mosaic, developmental overgrowth disorders. We recently demonstrated that the ‘hotspot’ variant PIK3CAH1047R exerts striking allele dose-dependent effects on stemness in human induced pluripotent stem cells (iPSCs), and moreover demonstrated multiple oncogenic PIK3CA copies in a substantial subset of human cancers. To identify the molecular mechanism underpinning PIK3CAH1047R allele dose-dependent stemness, we profiled isogenic wild-type, PIK3CAWT/H1047R and PIK3CAH1047R/H1047R iPSCs by high-depth transcriptomics, proteomics and reverse-phase protein arrays (RPPA). PIK3CAH1047R/H1047R iPSCs exhibited altered expression of 5644 genes and 248 proteins, whereas heterozygous hPSCs showed 492 and 54 differentially-expressed genes and proteins, respectively, confirming a nearly deterministic phenotypic effect of homozygosity for PIK3CAH1047R. Pathway and network-based analyses predicted a strong association between self-sustained TGFb/NODAL signaling and the ‘locked’ stemness phenotype induced by homozygosity for PIK3CAH1047R. This stemness gene signature was maintained without exogenous NODAL in PIK3CAH1047R/H1047R iPSCs and was reversed by pharmacological inhibition of TGFb/NODAL signaling but not by PIK3CA-specific inhibition. Analysis of PIK3CA-associated human breast cancers revealed increased expression of the stemness markers NODAL and POU5F1 as a function of disease stage and PIK3CAH1047R allele dosage. Together with emerging realization of the link between NODAL re-expression and aggressive cancer behavior, our data suggest that TGFb/NODAL inhibitors warrant testing in advanced breast tumors with multiple oncogenic PIK3CA copies.
2021-03-03 | PXD014719 | Pride
Project description:The human oncogene PIK3CA is frequently mutated in human cancers. The two hotspot mutations in PIK3CA, E545K and H1047R, have been shown to regulate widespread signaling events downstream of AKT. However, the impact of these activating mutations on the tyrosine phosphorylation signaling in the cell has not been studied. Here, we performed a global phosphotyrosine profiling using isogenic knockin cell lines containing these activating mutations. We identified 824 unique phosphopeptides from 308 proteins. We found a surprisingly widespread modulation of tyrosine phosphorylation levels of proteins in the knockin mutant cells, with many of the regulated proteins involved in important biological processes, including those in the cytoskeletal migration pathways and kinase regulated signaling. We observed a widespread modulation of the tyrosine kinome, with 24 of the tyrosine kinases showing either upregulation or downregulation in phosphorylation levels. Many of the regulated phosphosites that we identified were located in the kinase domain or the canonical activating sites, indicating that the kinases were active and hence their downstream signaling pathways. Our study thus shows that the activating mutations in PIK3CA result in widespread tyrosine signaling regulation, in addition to the serine/threonine signaling pathways activated by the canonical PI3K-AKT axis.
2018-10-24 | PXD001460 | Pride
Project description:The RTK/RAS/PI3K pathway is a key driver of tumorigenesis across all cancers, with 90% of Glioblastoma (GBM) tumors exhibiting alterations in this pathway. Among the specific genes in this pathway mutations in PI3K’s catalytic subunit, PIK3CA, are found in 11% of GBM tumors. Sequencing of GBM samples has revealed several known hotspot mutations that drive tumorigenesis in several cancers (E545K, H1047R), as well as a series of 63 in frame mutations (as indexed in COSMIC) that remain largely unclassified. To decode which of these PIK3CA variants function as drivers of GBM, we established an in vivo complementation screening platform for GBM. Our mouse GBM model relies on in utero electroporation (IUE) and CRISPR-Cas9 mediated knockout of NF1, p53, and PTEN (termed 3xCR). Because PTEN catalyzes the reverse reaction of PIK3CA, phosphorylation of PIP2 to PIP3, we complemented loss of PTEN with overexpression of E545K or H1047R, two bona fide hot spot driver mutations of PIK3CA. Both these variants accelerated tumor associated death, demonstrating that known PIK3CA drivers can complement PTEN loss in our system.
2019-10-09 | GSE123519 | GEO
Project description:We used CRISPR/Cas9 to knock in the cancer "hotspot" mutation PIK3CA-H1047R into one or both alleles of a wild-type induced pluripotent stem cell (iPSC) line (WTC11; Coriell # GM25256; P37-P38). Three clones from each genotype (wild-type, heterozygous, homozygous) were subjected to single-end mRNA sequencing (mean read depth per sample: 20 million) to determine whether PIK3CA-H1047R exerts allele dose-dependent transcriptional effects. Multidimensional scaling demonstrated distinct transcriptomic signatures of wild-type, heterozygous and homozygous cells. The transcriptome of heterozygous cells was nearly identical to wild-type controls, with only 131 differentially-expressed transcripts (FDR = 0.05). In contrast, homozygosity for PIK3CA-H1047R led to differential expression of 1,914 genes. This indicates widespread transcriptional remodeling with a sharp allele dose-dependency, suggestive of a threshold effect.
2019-02-15 | GSE126562 | GEO
Project description:We used CRISPR/Cas9 to knock in the cancer "hotspot" mutation PIK3CA-H1047R into one or both alleles of a wild-type induced pluripotent stem cell (iPSC) line (WTC11; Coriell # GM25256; P37-P38). Four cultures from each genotype (3 wild-type clones, 3 heterozygous clones, 2 homozygous clones) were subjected to paired-end mRNA sequencing (mean read length = 150 bp). The aim of this experiment was to confirm and expand upon previous transcriptomic results suggesting a near-binary transcriptional effect in homozygous versus heterozygous PIK3CA-H1047R iPSCs (publication doi: 10.1073/pnas.1821093116). According to the high-depth transcriptomic dataset, PIK3CA-H1047R/H1047R iPSCs exhibited altered expression of 5644 genes, whereas heterozygous hPSCs showed 492 differentially-expressed genes, supporting a nearly deterministic phenotypic effect of homozygosity for PIK3CA-H1047R. The differential gene expression analyses were performed based on the limma/voom/eBayes framework (doi: 10.1093/nar/gkv007), using customised scripts with FDR < 0.05 and absolute log2(fold-change) cut-off >= 1.3.
2019-10-01 | GSE134076 | GEO
Project description:Spatial Transcriptomics Using Multiplexed Deterministic Barcoding in Tissue
| PRJNA857431 | ENA
Project description:We present SIGNAL-seq (Split-pool Indexing siGNalling AnaLysis by sequencing): a multiplexed split-pool combinatorial barcoding method that simultaneously measures RNA and post-translational modifications (PTMs) in fixed single cells from 3D solid-tumour models. SIGNAL-seq PTM measurements are equivalent to mass cytometry and RNA gene detection is analogous to split-pool barcoding scRNA-seq. By measuring both mRNA ligand-receptor pairs and PTMs in single cells, SIGNAL-seq simultaneously reveals both inter- and intra-cellular signalling in tumour microenvironment organoids.
2024-02-24 | GSE256404 | GEO
Project description:We present SIGNAL-seq (Split-pool Indexing siGNalling AnaLysis by sequencing): a multiplexed split-pool combinatorial barcoding method that simultaneously measures RNA and post-translational modifications (PTMs) in fixed single cells from 3D solid-tumour models. SIGNAL-seq PTM measurements are equivalent to mass cytometry and RNA gene detection is analogous to split-pool barcoding scRNA-seq. By measuring both mRNA ligand-receptor pairs and PTMs in single cells, SIGNAL-seq simultaneously reveals both inter- and intra-cellular signalling in tumour microenvironment organoids.
2024-02-24 | GSE256403 | GEO
Project description:We present a new approach, Light-Seq, for multiplexed spatial indexing of intact biological samples using light-directed DNA barcoding in fixed cells and tissues followed by ex situ sequencing. Light-Seq combines spatially-targeted, rapid photocrosslinking of DNA barcodes onto cDNAs in situ with a novel one-step DNA stitching reaction to create pooled, spatially-indexed sequencing libraries. This light-directed barcoding enables in situ selection of multiple cell populations in intact fixed tissue samples for full transcriptome sequencing based on location, morphology, or protein stains, without cellular dissociation. Applying Light-Seq to mouse retinal sections, we recovered thousands of differentially enriched transcripts from three adjacent cellular layers and discovered new biomarkers for a very rare neuronal subtype, dopaminergic amacrine cells, from only 4-8 individual cells per section. Light-Seq provides an accessible workflow to combine in situ imaging and protein staining with next-generation sequencing of the same cells, leaving the sample intact sample for further analysis post-sequencing.
2022-10-10 | GSE208650 | GEO