Project description:This study examined the effect of mutant PIK3CAH1047R expression in mammary subsets of preneoplastic mammary glands from K8-creERT2/PIK3CA H1047R mice Mammary cell subpopulations were isolated from K8-creERT2/PIK3CAH1047R and K8-creERT2 control animals 4 weeks after activation of PIK3CA H1047R transgene expression by Tamoxifen injection. Pooled mammary glands of 2-3 estrus-synchronized mice per genotype were sorted in 3 independent sortings and used for microarray analysis (20 samples in total).
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.
Project description:This study examined the effect of mutant PIK3CAH1047R expression in mammary subsets of preneoplastic mammary glands from Lgr5-creERT2/PIK3CA H1047R mice Mammary cell subpopulations were isolated from Lgr5-creERT2/PIK3CA H1047R and Lgr5-creERT2 control animals 4 weeks after activation of PIK3CA H1047R transgene expression by Tamoxifen injection. Pooled mammary glands of 2-3 estrus-synchronized mice per genotype were sorted in 3 independent sortings and used for microarray analysis (24 samples in total).
Project description:This study examined the effect of mutant PIK3CAH1047R expression in mammary subsets of preneoplastic mammary glands from Lgr5-creERT2/PIK3CA H1047R mice
Project description:This study examined the effect of mutant PIK3CAH1047R expression in mammary subsets of preneoplastic mammary glands from K8-creERT2/PIK3CA H1047R mice
Project description:MCF10A cells: control vs. PIK3CA mutant (H1047R) Transcriptional profiling of MCF10A comparing control (expressing JP1520-PIK3CA-WT; Addgen plasmid #14570) and PIK3CA mutant (JP1520-PIK3CA-H1047R; Addgene plasmic#14572). Goal was to determine the effects of the PIK3CA H1047R mutation in the on global gene expression in MCF10A cells.
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).
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.