Plasma membrane proteomics identifies Notch1 as a potential regulator of ras-induced senescence

Abstract

BackgroundOncogene-induced senescence (OIS) is an intrinsic tumour suppressor mechanism leading to stable cell-cycle arrest in response to oncogene activation. OIS is a heterogeneous phenotype of multiple effector mechanisms; understanding of in-vivo OIS is lacking because of the difficulty in identifying senescence. We wished to establish a cell-surface phenotype of OIS. MethodsWe used ER:Ras IMR90 human diploid fibroblasts, which undergo OIS after 6 days with 4-hydroxytamoxifen (4-OHT). We used SILAC (stable isotope labelling with amino acids in cell culture)-based proteomics and three labelling conditions (light, IMR90 plus 4-OHT; medium [lysine, K+4Da, arginine, R+6Da] ER:ras IMR90; heavy [K+8Da, R+10Da] ER:Ras IMR90 plus 4-OHT), combined with cell surface aminooxybiotinylation before streptavidin pulldown. Tryptic peptides were fractionated by high pH reversed-phase high performance liquid chromatogrpahy and then subjected to liquid chromatograph mass spectrometry. Data were processed by the analytical packages MaxQuant and MASCOT. Hits were validated by fluorescence-activated cell sorting (FACS), quantitative PCR, and immunoblotting. shRNA-mediated knockdown of protein expression used the murine stem cell virus-miR30 system. Findings899 proteins were identified, of which 73% were present at the cell surface by Gene Ontology annotation. Notch1 was significantly upregulated in OIS compared with both control conditions (3·1–3·4 fold). Upregulation was confirmed by both FACS and immunoblotting. Downstream Notch target-genes were also upregulated in OIS. Treatment with the γ-secretase inhibitor DAPT increased both senescence-associated heterochromatin foci (SAHF) and senescence-associated β galactosidase (SA-β-gal) activity, features of OIS. However, Notch1 knockdown reduced both SAHF and SA-β-gal. The effect of DAPT was not mediated through canonical Notch1 signalling since RBP-j knockdown had no effect upon DAPT-mediated SAHF and SA β-gal upregulation. Numb, a canonical Notch pathway inhibitor, is upregulated in OIS. Numb may underpin a transition switch from canonical to non-canonical Notch signaling in OIS. InterpretationPlasma membrane proteomics has identified a cell-surface phenotype of OIS. Notch 1 cell surface expression and downstream target-genes are upregulated in OIS. The transition to OIS is correlated with a transition from canonical to non-canonical Notch1 signalling that may be driven by Numb expression. FundingCancer Research UK.