An RNA interference screen for identifying downstream effectors of the p53 and pRB tumour suppressor pathways involved in senescence

Abstract

BackgroundCellular senescence is an irreversible cell cycle arrest that normal cells undergo in response to progressive shortening of telomeres, changes in telomeric structure, oncogene activation or oxidative stress and acts as an important tumour suppressor mechanism.ResultsTo identify the downstream effectors of the p53-p21 and p16-pRB tumour suppressor pathways crucial for mediating entry into senescence, we have carried out a loss-of-function RNA interference screen in conditionally immortalised human fibroblasts that can be induced to rapidly undergo senescence, whereas in primary cultures senescence is stochastic and occurs asynchronously. These cells are immortal but undergo a rapid irreversible arrest upon activation of the p53-p21 and p16-pRB pathways that can be readily bypassed upon their inactivation. The primary screen identified 112 known genes including p53 and another 29 shRNAmirs targetting as yet unidentified loci. Comparison of these known targets with genes known to be up-regulated upon senescence in these cells, by micro-array expression profiling, identified 4 common genes TMEM9B, ATXN10, LAYN and LTBP2/3. Direct silencing of these common genes, using lentiviral shRNAmirs, bypassed senescence in the conditionally immortalised cells.ConclusionThe senescence bypass screen identified TMEM9B, ATXN10, LAYN and LTBP2/3 as novel downstream effectors of the p53-p21 and p16-pRB tumour suppressor pathways. Although none of them has previously been linked to cellular senescence, TMEM9B has been suggested to be an upstream activator of NF-κB signalling which has been found to have a causal role in promoting senescence. Future studies will focus on determining on how many of the other primary hits also have a casual role in senescence and what is the mechanism of action.

Cell-based screen for altered nuclear phenotypes reveals senescence progression in polyploid cells after Aurora kinase B inhibition

“…, 2008 ; Leal et al. , 2008 ; Rovillain et al. , 2011 ), attempts at “senescence-inducing” screens are limited ( Ewald et al.…”

A high-content cellular senescence screen identifies candidate tumor suppressors, including EPHA3

“…Our screen contrasts with previous studies, which focused on senescence bypass events, and biased for discovery of genes intrinsically linked to or required for the specific arrest pathway 11 - 13 . Furthermore, the proposed pro-tumorigenic effect of a senescence-associated inflammatory response 14 warrants a closer study to assess whether senescence can be induced upon loss of relevant gene function.…”

Tumour suppressors and cellular senescence

“…One of the key activators of the SASP is the nuclear factor kappa‐light‐chain‐enhancer of activated B‐cells (NF‐κB) family of ubiquitously expressed transcription factors, important for the cellular response to stress . This family of proteins regulate innate and adaptive immunity, which has a pro‐tumourigenic side effect ; however, they have recently been shown to promote entry into senescence . These opposite effects of NF‐κB may depend on a variety of factors, including the subunit expressed, the particular biological context and the nature of the upstream signal, which leads to activation of this pathway .…”