RNA-Seq Analysis Reveals a Transcriptional Feedback Loop in EGF-PIP3 Signalling

Presentation given at the Babraham Institute talks, Cambridge, UK

Transcript

1. RNA-Seq analysis reveals a transcriptional feedback loop in EGF-PIP3 signalling Vladimir Kiselev Babraham Institute Talks 23/05/14
2. EGF-PIP3 signalling PIP3 PIP2 PI3K* PTEN PI3K EGFR* EGFR EGF Growth Proliferation Survival Motility …
3. EGF-PIP3 signalling PIP3 PIP2 PI3K* PTEN p110α H1047R oncomutant (1 WT allele, 1 mutant allele) PTEN KO (2 mutant alleles) Growth Proliferation Survival Motility … in cancer
4. Experimental design Cellular! models of! MCF10a WT Isogenic MCF10a p110α H1047R oncomutant (1 WT allele, 1 mutant allele) Isogenic MCF10a PTEN KO (2 mutant alleles) Day 1:! Seed cells Day 2:! Starve cells! for 18 hours WT + p110α selective inhibitor (A66) Day3 -20 15 40 90 180 300 Time, min A66 EGF stimulation RNA-Seq x3 Veronique Juvin no EGF
5. Effect of constitutive mutations regardless EGF PI3K+ PTEN−/− 1809 511 474 • Differential expression before stimulation (t = 0) • p-value < 0.01 • No effect of A66 treatment • 474 genes identified
6. EGF treatment effect • Differential expression upon EGF stimulation in WT (from 0 min to 300 min) • p-value < 0.01 • 249 of genes affected by both mutations are also affected by EGF in WT PI3K+ & PTEN−/− EGF (WT) 4964 225 249
7. Signal transduction diagram PI3K+ & PTEN-/- EGF stimulation TFs TFs TFs Genes Genes Genes
8. Biological process Multicellular organismal development Developmental process Anatomical structure development System development Anatomical structure morphogenesis Organ development Cardiovascular system development Blood vessel morphogenesis Angiogenesis 97 23 89 70 26 95 60 Locomotion Chemotaxis Neutrophil chemotaxis Leucocyte chemotaxis Immune system process Cell adhesion Response to external stimulus Response to stress Response to wounding 30 33 40 33 21 6 GO enrichment analysis Genes Genes
9. PI3K+ & PTEN-/- EGF stimulation TFs TFs Genes Genes RNA-Seq ISMARA What about TFs? Integrated System for Motif Activity Response Analysis
10. ISMARA workflow Database of TFBM ~ Activity of every TFBM in every sample RNA-Seq Promoter-motif matrix Promoter-sample matrix
11. PI3K+ & PTEN-/- EGF stimulation TFs TFs Genes Genes Differential activity of TF binding motifs Activity score Gene number PRDM1 TFBM TFBMs TFBMs = But activities are different!
12. Targets of PRDM1 PI3K+ & PTEN-/- EGF stimulation TFs TFs Genes Genes PIK3IP1! negative regulator of hepatic PI3K activity! GAB2! involved in the activation of PI3K! DENND2D! GEF, tumour suppressor in lung cancer, PI3K sensitive! LPXN! interacts with tyrosine kinases ! (upstream of PI3K)! VWA5A! breast cancer suppressor! TACC2! tumor suppressor
13. Transcriptional feedback loop! PI3K+ & PTEN-/- EGF stimulation TFs TFs TFs Genes Genes Genes
14. Acknowledgements • Le Novere lab: Nicolas Le Novere, Marija Jankovic, Pnar Pir, Martina Froehlich, Nicolas Rodriguez, Massimo Lai, Lu Li • Stephens/Hawkins labs: Len Stephens, Phil Hawkins, Veronique Juvin, Mouhannad Malek • Luscombe lab: Nicholas Luscombe, Kathi Zarnack, Raphaelle Luisier, Filipe Tavares-Cadete, Bori Mifsud, Anna Poetsch, Alessandra Vigilante, Elodie Darbo, Robert Sugar, Inigo Martincorena, Juanma Vaquerizas, Judith Zaugg • BI bioinformatics group: Simon Andrews, Anne Segonds-Pichon, Phil Ewels, Felix Krueger • Others: Sven Bergmann, Mikhail Spivakov