Gene expression profiling of HUVEC-s expressing PIK3CA with H1047R point mutation
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE196311 | GEO | 2023/06/10
REPOSITORIES: GEO
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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.
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Project description:Gene targeting was carried out by Taconic Artemis (Cologne, Germany) to introduce a point mutation resulting in conversion of CAT (H) to CGT (R) in the C-terminal p110α kinase domain, and a neo selection cassette flanked by frt sites (publication doi: 10.1038/s41467-017-02002-4). MEFs were made from PIK3CA-H1047R/WT and PIK3CA-WT/WT E13.5 embryos (4 independent MEFs per genotype), treated with 1 μM 4-OHT for one day and gathered 48h after the start of 4-OHT induction were subjected to mRNA sequencing to determine changes in gene expression upon short term PIK3CA activation in the absence of other transforming mutations. PIK3CA-H1047R/WT MEFS exhibited altered expression of 71 genes (q cutoff <0.05).
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Project description:We have compared the proteome, transcriptome and metabolome of two isogenic cell lines: MCF-10A, derived from human breast epithelium, and the mutant MCF-10A-H1047R. These cell lines differ by a single amino acid substitution (H1047R) caused by single nucleotide change in one allele of the PIK3CA gene which encodes the catalytic subunit p110α of phosphatidylinositol 3-kinase (PI3K). The H1047R mutation of PIK3CA is one of the most frequently encountered somatic cancer-specific mutations. In MCF-10A, this mutation induces an extensive cellular reorganization that far exceeds the known signaling activities of PI3K. The changes are highly diverse; with examples in structural protein levels, the DNA repair machinery and sterol synthesis. Gene set enrichment analysis reveals a highly significant concordance of the genes differentially expressed in MCF-10A-H1047R cells and the established protein and RNA signatures of basal breast cancer. No such concordance was found with the specific gene signatures of other histological types of breast cancer. Our data document the power of a single base mutation, inducing an extensive remodeling of the cell toward the phenotype of a specific cancer. 2 cell lines (H1047R and WT), 4 time points (0, 6, 12, 24 hours), 3 replicates
2015-01-19 | E-GEOD-63452 | biostudies-arrayexpress
Project description:We have compared the proteome, transcriptome and metabolome of two isogenic cell lines: MCF-10A, derived from human breast epithelium, and the mutant MCF-10A-H1047R. These cell lines differ by a single amino acid substitution (H1047R) caused by single nucleotide change in one allele of the PIK3CA gene which encodes the catalytic subunit p110α of phosphatidylinositol 3-kinase (PI3K). The H1047R mutation of PIK3CA is one of the most frequently encountered somatic cancer-specific mutations. In MCF-10A, this mutation induces an extensive cellular reorganization that far exceeds the known signaling activities of PI3K. The changes are highly diverse; with examples in structural protein levels, the DNA repair machinery and sterol synthesis. Gene set enrichment analysis reveals a highly significant concordance of the genes differentially expressed in MCF-10A-H1047R cells and the established protein and RNA signatures of basal breast cancer. No such concordance was found with the specific gene signatures of other histological types of breast cancer. Our data document the power of a single base mutation, inducing an extensive remodeling of the cell toward the phenotype of a specific cancer.
2015-01-19 | GSE63452 | 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.
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Project description:Human HCC cell lines SNU387 and SNU449 stably transfected (lentivirus) with PIK3CA E545K, PIK3CAH1047R, EGFP or WT. Comparison to determine differential effectors of PIK3CA helical and kinase domain mutants by microarray.
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Project description:We wanted to know how introduction of H1047R oncogenic mutation of PIK3CA (p110a subunit of PI3K) in HER2-overexpressing MCF10A human non-transformed mammary epitheial cell lines introduces change in gene expression levels. Our control was HER2-overexpressing MCF10A cell line with wild-type (WT) PIK3CA. Mutated oncogene-induced gene expression signature was measured and compared with that induced by the WT oncogene from cell lines harvested at 70-80% confluency.
2021-03-16 | GSE117389 | GEO
Project description:Gene expression microarray analyses of PIK3CA H1047R and E545K induced preneoplastic lesions and tumors compared to age matched controls. Plasmids expressing oncogenic forms of PIK3CA (H1047R or E545K mutants) were delivered to the mouse liver by tail vein hydrodynamic injection combined with Sleeping Beauty–mediated somatic integration (SBT-HTVI). Resulting preneoplastic and neoplastic lesions were subjected to gene expression microarray analysis. The putative PIK3CA target gene Galectin1 (Gal1) was further characterized by in vitro studies. Transfection of either PIK3CA E545K or H1047R mutants via SBT-HTVI was sufficient to induce hepatocellular carcinomas in mice. A stepwise hepatocarcinogenesis from singular pericentral lipid-rich preneoplastic hepatocytes to clusters, expansive preneoplastic lesions, and HCCs was observed. In PIK3CA H1047R injected mice, HCCs were detected in the 12 months injection group, while PIK3CA E545K injections resulted in tumor occurrence as early as 3 months after SB-HTVI. Histologically, a predominant lipid-rich phenotype characterized all tumorigenesis stages, as confirmed by Sudan III staining and electron microscopy. Immunohistochemically, preneoplastic lesions and tumors displayed high levels of the lipid master regulators Fatty acid synthase and Stearoyl-CoA desaturase-1. Gal1 was commonly overexpressed in PIK3CA-driven preneoplastic and neoplastic liver lesions by gene expression microarray analysis compared with age-matched controls.
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Project description:Gain-of-function mutation of PIK3CA represents one of the most common oncogenic events in human malignancy, making PI3K an attractive target for cancer therapy. Despite the great promise of targeted therapy, drug resistance is likely to develop, causing treatment failure. To elucidate resistance mechanisms to PI3K-targeted therapy, we constructed a mouse model of breast cancer conditionally expressing PIK3CA-H1047R. Surprisingly, the majority of mammary tumors induced by PIK3CA-H1047R expression recurred following PIK3CA-H1047R inactivation. Genomic analyses of recurrent tumors revealed multiple lesions, including spontaneous focal amplification of c-Met or c-Myc. While amplification of c-Met allowed tumor survival dependent on activation of endogenous PI3K, tumors with amplification of c-Myc become independent of the PI3K pathway. Functional analyses further demonstrated that c-Myc contributed to tumors’ independence of oncogene and resistance to PI3K inhibition. Together, our data suggest that MYC elevation in tumors may be a potential mechanism conferring resistance to current PI3K-targeted therapies. Affymetrix SNP array analysis was performed with Mouse Diversity Genotyping Arrays (Affymetrix) on genomic DNA extracted from frozen biopsies of 6 recurrent mouse mammary tumor samples. Copy number analysis was performed for the mouse mammary tumors using genomic DNA from normal mammary tissue as the reference for copy number inference.
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