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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Endogenous expression of an oncogenic PI3K mutation leads to activated PI3K pathway signaling and an invasive phenotype

ABSTRACT: The PTEN/PI3K pathway is commonly mutated in cancer and therefore represents a rational target for therapeutic intervention. In order to investigate the primary phenotype(s) mediated by mutant PIK3CA in a clean and highly patient-relevant context, we utilized a non-tumorigenic MCF10A parental and isogenic knock-in cell line that harbors a common activating PIK3CA kinase domain mutation (H1047R). We found that introduction of an endogenously mutated PIK3CA primarily results in a marked epithelial-mesenchymal transition (EMT) and invasive phenotype compared to isogenic wild-type cells. Moreover, a potent and selective inhibitor of PIK3CA (GDC-0941) was highly effective and selective on reversing this phenotype compared to cell-proliferation, highlighting a potential new paradigm for studying PI3K-pathway targeted agents. Keywords: Expression Array Gene expression profiles from a parental MCF10A breast line and an MCF10A clone that has a PI3K activating mutation (H1047R) knocked-in. We plated the cells in 10cm dishes overnight in media with serum, but lacking EGF or insulin. DMSO or GDC-0941 (PI3K inhibitor) at an EC50 concentration were added to triplicate plates of cells in the morning. After 4 hours the RNA was harvested using the Qiagen RNeasy kit. There are 12 samples total.

ORGANISM(S): Homo sapiens

SUBMITTER: Peter Haverty

PROVIDER: E-GEOD-17785 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Endogenous expression of an oncogenic PI3K mutation leads to activated PI3K pathway signaling and an invasive phenotype

Project description:The PTEN/PI3K pathway is commonly mutated in cancer and therefore represents a rational target for therapeutic intervention. In order to investigate the primary phenotype(s) mediated by mutant PIK3CA in a clean and highly patient-relevant context, we utilized a non-tumorigenic MCF10A parental and isogenic knock-in cell line that harbors a common activating PIK3CA kinase domain mutation (H1047R). We found that introduction of an endogenously mutated PIK3CA primarily results in a marked epithelial-mesenchymal transition (EMT) and invasive phenotype compared to isogenic wild-type cells. Moreover, a potent and selective inhibitor of PIK3CA (GDC-0941) was highly effective and selective on reversing this phenotype compared to cell-proliferation, highlighting a potential new paradigm for studying PI3K-pathway targeted agents. Keywords: Expression Array

2011-08-10 | GSE17785 | GEO

Transcriptional landscape of oncogenic PI3K

Project description:RNA-seq performed in triplicates in MCF10A and MEF cells. Wildtype/parental vs PIK3CA H1047R mutant. MEFs were treated with DMSO or GDC0032 (500nM) PI3Ka inhibitor treatment while MCF10A cells were treated with DMSO or alpelisib (BYL719) at 1uM.

2022-04-25 | GSE157858 | GEO

Pentraxin-3 is a PI 3-Kinase Effector that Regulates Stem-Cell-Like Traits in Basal-Like Breast Cancer

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.

2017-02-28 | GSE93422 | GEO

Transcriptomic effect of retroviral overexpression of PIK3CA-H1047R in breast epithelial MCF10A cells; +/- 500 nM BYL719 as a function of time (48h and 120h).

Project description:The aim of this experiment was to evaluate the transcriptomic effects of PI3K pathway hyperactivation vs inhibition, in the context of PIK3CA-H1047R overexpression in MCF10A breast epithelial cells. The cells were processed for RNA sequencing, with the t values from subsequent differential gene expression processing used for gene set enrichment analysis (GSEA) with the MSigDB HALLMARK_PI3K_AKT_MTOR_SIGNALING gene signature. We demonstrate that the HALLMARK_PI3K_AKT_MTOR_SIGNALING gene signature can be used as a transcriptional footprint of PI3K pathway activation: it correctly predicts activation of the pathway in DMSO-treated PIK3CA-H1047R MCF10A cells relative to EV controls; conversely, it also correctly predicts pathway inhibition upon p110 inhibition with BYL719.

2021-09-20 | GSE184277 | GEO

Development of gene expression signatures for MCF10A cell lines carrying HER2 and WT or H1047R PIK3CA oncogenes.

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

Knock-in of PIK3CA-H1047R into MCF-10A

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 p110Î± 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

Oncogenic PI3K mutations lead to NF-kB-dependent cytokine expression following growth

Project description:While growth factor-independent signaling and proliferation are well-established hallmarks of cancer, little is known regarding growth factor-independent changes in gene expression which occur downstream from oncogenes. The PI3K pathway is one of the most commonly misregulated signaling pathways in human cancers. Here, MCF10A cells expressing the two most common PI3K mutations, PIK3CA E545K and H1047R, were used to identify the repertoire of genes altered by oncogenic PI3K mutations following growth factor deprivation. This gene set most closely correlated with gene signatures from claudin-low and basal-like breast tumors, and categorical enrichment analyses suggested that NF-kB target genes were dramatically upregulated by these mutations. An IKKb inhibitor was used to identify the subset of PI3K-driven genes that is NF-kB dependent. Interestingly, virtually all of these NF-kB dependent genes were secreted proteins, suggesting a paracrine role for this gene set. Among these genes was IL-6, a cytokine frequently expressed in tumors which plays a critical role in generating a tumor-promoting microenvironment. Consistent with this, conditioned media from cells expressing the E545K or H1047R mutations led to increased STAT3 activation in recipient THP-1 monocytes or normal epithelial cells in a NF-kB and IL-6-dependent manner. Together, these data describe a PI3K-driven, NF-kB-dependent gene expression profile which may play a critical role in promoting a microenvironment amenable to tumor progression. 39 normal cell lines vs treated cell lines for micorarray analysis

2012-04-30 | E-GEOD-33403 | biostudies-arrayexpress

High-dose oncogenic PIK3CA drives constitutive cellular stemness through increased NODAL/TGFb signaling

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

Activating Mutations in PIK3CA result in Widespread Modulation of the Tyrosine Phosphoproteome

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

Global proteomic alterations in extracellular vesicles induced by oncogenic PIK3CA mutations

Project description:MCF10A cells, and two isogenic mutant cell lines with known mutations in PIK3CA (E545 K and H1047R) were cultured in triplicate using culture medium depleted of extracellular vesicles. Cell culture supernatants were collected to study the extracellular vesicles (EVs) released by these cells. EVs were isolated from the samples by ultracentrifugation and their proteins digested. The resulting peptides were labeled with TMT and analyzed by mass spectrometry to identify differentially expressed proteins.

2023-10-22 | PXD031718 | Pride

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