Unknown,Transcriptomics,Genomics,Proteomics

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Differential accumulation of splice variants and transcripts as a result of PI3K inhibition in T lymphocytes and the potential role of their gene products in T cell silencing

ABSTRACT: Using measles virus induced T cell suppression as a model, we established that T cell inhibitory protein isoforms can be produced from alternatively spliced pre-mRNAs as a result of virus-mediated ablation of T cell receptor dependent activation of the phosphatidylinositol-3-kinase (PI3K). To asses production of alternative splice variants in response to PI3K abrogation in T cells at a whole cell level, we performed a Human Exon 1.0 ST Array on RNAs isolated from T cells stimulated only or stimulated after PI3K inhibition. We developed a simple algorithm based on a splicing index to detect genes that undergo alternative splicing (AS) or are differentially regulated (RG) on T cell suppression. Applying our algorithm on this model 9% of the genes were assigned as AS, while only 3% were attributed to RG. Though there are overlaps, AS and RG genes differed with regard to functional regulated at the level of AS or RG were found enriched in different functional groups with AS targeting e. g. extra cellular matrix (ECM)-receptor interaction and focal adhesion, while cytokine-receptor interaction, Jak-STAT and p53 pathways were mainly RG. When combined, AS/RG dependent alterations targeted pathways essential for T cell receptor signaling, cytoskeletal dynamics and cell cycle entry strongly supporting the notion that PI3K abrogations interferes with key T cell activation processes at both levels, and that candidates represented within both categories bear the potential to actively contribute to T cell suppression total samples analysed are 8: 4 stimulated and 4 stimulated and inihibited from 4 donors

ORGANISM(S): Homo sapiens

SUBMITTER: Susanne Kneitz

PROVIDER: E-GEOD-38255 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Activating mutations of PIK3CA are the most frequent genomic alterations in estrogen receptor (ER)-positive breast tumors and selective PI3Kα inhibitors are in clinical development. The activity of these agents, however, is not homogenous and only a fraction of patients bearing PIK3CA-mutant ER-positive tumors benefit from single agent administration. Searching for mechanisms of resistance, we observed that suppression of PI3K signaling with different agents results in induction of ER-dependent transcriptional activity as demonstrated by changes in expression in genes containing ER binding sites, enhanced ER transcription and increased occupancy by the ER of promoter regions of upregulated genes. Furthermore, expression of ESR1 mRNA and ER protein levels themselves were also increased upon PI3K inhibition. These changes in gene expression were confirmed in vivo in xenograft and patient derived models and in tumors from patients undergoing treatment with the PI3Kα inhibitor BYL719. The observed effects on transcription were enhanced by the addition of estradiol and suppressed by the anti-ER therapies fulvestrant and tamoxifen. Fulvestrant markedly sensitized ER-positive tumors to PI3Kα inhibition. We propose that increased ER transcriptional activity may be a compensatory mechanism that limits the activity of PI3K inhibitors and that combined PI3K and ER inhibition is a rational approach to target these tumors. The aim of our study was to explore the mechanism by which combination of PI3K pathway inhibitors and estrogen receptor function blockade results in superior antitumor activity. We aimed to evaluate whether changes in ER function were influencing the clinical response to anti-PI3K therapy in ER-positive breast tumors that harbor PI3K pathway activation. For this purpose, we planned to use various specific PI3K inhibitors, namely: BYL719 (p110α specific catalytic inhibitor), GDC0941 (pan-PI3K inhibitor), GDC0032 and BAY80-6946 (p110sparing PI3K inhibitors) in a panel of ER-positive breast cancer cell lines and xenografts that harbor PIK3CA activating mutations. We also used MK2206 (pan-AKT allosteric inhibitor) to inhibit the PI3K pathway in ER-positive cell lines which activate this pathway through PTEN loss. Finally, in order to evaluate the role of ER up-regulation as a pro-survival signal in our in vitro and in vivo models, we planned to use the selective ER modulator 4-hydroxy-tamoxifen (4-OHT) and degrader fulvestrant. For the in vivo experiments, the number of animals in each group was calculated to measure a 25% difference between the means of placebo and treatment groups with a power of 80% and a p value of 0.01. Host mice carrying xenografts were randomly and equally assigned to either control or treatment groups. Animal experiments were conducted in a controlled and non-blinded manner. Moreover, we evaluated by means of RNAseq gene expression changes breast cancer patients that underwent BYL719 based therapy to validate our in vitro findings in terms of ER expression. In vitro experiments were performed at least two times and at least in triplicate for each replica.

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Differential accumulation of splice variants and transcripts as a result of PI3K inhibition in T lymphocytes and the potential role of their gene products in T cell silencing

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