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

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. Overall design: Transcriptomic data from untransformed, immortalised human breast epithelial MCF10A cells with retrovirally-mediated overexpression of the PIK3CA-H1047R oncogene, alongside the respective empty vector (EV) control cells; assessed in growth factor-replete conditions, with PIK3CA-H1047R also exposed to either DMSO or 500 nM BYL719 (p110alpha-specific inhibitor) in order to determine the extent to which the transcriptomic phenotype can be reversed. Data were obtained from two treatment time points - 48h and 120h. Following differential gene expression analysies with limma-voom, the genes were ranked according to the t values specifying a given comparison, and used to determine whether conventional gene set enrichment analysis (GSEA) with the the mSigDb "HALLMARK_PI3K_AKT_MTORC1" gene signature would distinguish samples with PI3K pathway activation vs inhibition.