Oncogene (2008) 27, 1961–1970 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE Cellular senescence bypass screen identifies new putative tumor suppressor genes JFM Leal1, J Fominaya1, A Casco´ n2, MV Guijarro1, C Blanco-Aparicio1, M Lleonart3, ME Castro1, SRamon y Cajal 3, M Robledo2, DH Beach4 and A Carnero1 1Experimental Therapeutics Programme, Centro Nacional de Investigaciones Oncolo´gicas (CNIO), Madrid, Spain; 2Molecular Pathology Programme, Centro Nacional de Investigaciones Oncolo´gicas (CNIO), Madrid, Spain; 3Departamento de Patologı´a, Hospital Vall d’Hebron, Barcelona, Spain and 4Institute for Cell and Molecular Sciences, Center for Cutaneous Biology, London, UK Senescence is a mechanism that limits cellular lifespan and Introduction constitutes a barrier against cellular immortalization. To identify new senescence regulatory genes that might play a Replicative senescence is characterized by a progressive role in tumorigenesis, we have designed and performed a loss of proliferative potential with the increase of popu- large-scale antisense-based genetic screen in primary lation doublings, resulting in a permanent and irrever- mouse embryo fibroblasts (MEFs). Out of this screen, sible cell-cycle arrest. Although the process of senescence we have identified five different genes through which loss occurs both in vitro and in vivo (Dimri et al., 1995; of function partially bypasses senescence. These genes Schmitt et al., 2002; Shay and Roninson, 2004; Braig belong to very different biochemical families: csn2 et al., 2005; Collado et al., 2005; Michaloglou et al., (component of the Cop9 signalosome), aldose reductase 2005), the transition to the senescent phenotype is (a metabolic enzyme) and brf1 (subunit of the RNA commonly studied in culture where a cell population polymerase II complex), S-adenosyl homocysteine hydro- can be grown and monitored. Replicative senescence can lase and Bub1. Inactivation, at least partial, of these genes be a consequence of shortening and dysfunction of the confers resistance to bothp53- and p16INK4a-induced telomeres, due to the inability of most human cells to proliferation arrest. Furthermore, such inactivation in- replicate the chromosomal ends (Serrano and Blasco, hibits p53 but not E2F1 transcriptional activity and 2001; Greider and Blackburn, 2004). Nevertheless, impairs DNA-damage-induced transcription of p21. Since cellular senescence (also including accelerated senes- the aim of the screen was to identify new regulators of cence) is a more general process not only triggered tumorigenesis, we have tested their inactivation in human by accumulation of cell divisions, but also by activation tumors. We have found, either by northern blot or of oncogenes (Serrano et al., 1997), DNA damage, epi- quantitative reverse transcriptase–PCR analysis, that genetic changes and oxidative stress (Campisi, 2001; the expression of three genes, Csn2, Aldose reductase Shay and Roninson, 2004). Senescent cells cannot be and Brf1, is lost at different ratios in tumors of different stimulated to re-enter the cell cycle by physiological origins. These genes are located at common positions of mitogens, become resistant to apoptosis and acquire loss of heterogeneity (15q21.2, 7q35 and 14q32.33); altered differentiated characteristics. Moreover, accele- therefore,we have measured genomic losses of these rated senescence driven by any of these stresses is inde- specific genes in different tumors. We have found that pendent of the telomeric length (Serrano and Blasco, Csn2 and Brf1 also show genomic losses of one allele in 2001; Suzuki et al., 2001) and is not prevented by expres- different tumors. Our data suggest that the three genes sion of telomerase (Morales et al., 1999). Regardless of identified in the genome-wide loss-of-function genetic the mechanism triggering cellular senescence, the signal- screen are putative tumor suppressors located at ing cascade leading to growth arrest seems to be similar 15q21.2; 7q35 and 14q32.33. and it involves the p53 and pRB pathways (Sherr and Oncogene (2008) 27, 1961–1970; doi:10.1038/sj.onc.1210846; McCormick, 2002). Overcoming the restriction that published online 29 October 2007 cellular senescence poses to immortalization may be a primary step during the transformation to malignancy Keywords: senescence; genetic screening; tumor sup- (Hanahan and Weinberg, 2000). pressor; p53; BRF1; Csn2 During immortalization, cells acquire genetic altera- tions that override the normal mechanisms controlling senescence (For review see Hanahan and Weinberg, 2000; Sherr and McCormick, 2002; Shay and Roninson, Correspondence: Dr A Carnero, Experimental Therapeutics 2004; Campisi, 2005). Among the alterations that Programme, Centro Nacional de Investigaciones Oncologicas (CNIO), immortalize cells, those that inactivate tumor suppressor c/Melchor Fernandez Almagro no3, Madrid 28029, Spain. E-mail: [email protected] genes in the p53 and pRB pathways are very frequent Received 20 June 2007; revised 31 August 2007; accepted 7 September in cancer. Tumor suppressor genes may get inactivated 2007; published online 29 October 2007 by either deletion of one or both alleles, promoter Cellular senescence bypass screen JFM Leal et al 1962 methylation, splice-site mutations and nonsense muta- fragments, we measured their efficacy at reducing the tions that induce premature translational termination protein levels of their respective targets. NIH3T3 cells and destabilize mRNA transcripts (or a combination carrying either of Csn2, ARase and BRF1 HA-tagged thereof). Such alterations result in a complete absence or proteins were infected with the correspondent antisense partial reduction of the tumor suppressor protein in the in the pMARXIV vector, and the effect of the antisense affected cells, conferring them selective advantage in was quantitated by western blotting. As shown in clonal selection for tumor progression. Figure 1a, the protein level of the different HA-tagged In this work, we designed and performed a genome- genes was reduced by average of 50%. Expression of wide loss-of-function genetic screen to identify addi- these antisense constructs partially bypasses growth tional putative tumor suppressor genes controlling arrest in senescence and is able to produce a moderate senescence that might act as tumor suppressors. increase in the lifespan of MEFs (Figure 1b and data not shown). In 3T3 experiments, the antisense fragments increased MEFs lifespan approximately four population doublings (Figure 1c), although the cells finally entered Results senescence. Moreover, the expression of the different antisense fragments also provides an early escape from Pre-senescent MEFs were infected with retrovirus senescence of the cultures. However, downregulation of carrying a library of senescent MEF’s transcriptome in these genes do not alter the p53 levels nor phosphoryla- antisense orientation (Carnero et al., 2000). MEFs were tion status in MEFs (Supplementary Materials 2). seeded at low concentration at doubling time of 10–12 In IMR90 human diploid fibroblasts, active shRNA and left to senesce. Clones that were able to grow were against Csn2 and ARase do not produce viable cell lines, identified, provirus recovered and sequenced. while active shRNA against BRF1 enhances the lifespan Out of our genome-wide loss-of-function screen, we in 6–8 population doublings causing cells to abruptly identified five different antisense fragments that targeted enter apoptosis (data not shown). proteins with very different biochemical properties (Table 1). Among them, an antisense against Csn2 (a component of the Cop9 signalosome) was recovered Effect of antisense on pRB and p53-dependent cell growth eight independent times. Antisense fragments against arrest Aldose reductase and S-adenosyl homocysteine hydro- To explore the possible mechanisms through which lase (two metabolic enzymes) and against BRF1 (a these genes might affect senescent arrest, we studied the subunit of the RNA polymerase III complex) were effect of the different antisense fragments on the p53 and recovered twice. Finally, an antisense against Bub1 was pRb checkpoints. First, we measured the ability of these recovered once. The size of the fragments varies from antisense constructs to bypass p53-induced arrest. To 130 to 210 nucleotides and they map to different regions this end, we expressed the different antisense fragments in the mRNAs, including the 30 untranslated region, as in a cell line derived from p53-null MEFs expressing the in the case of Csn2 and Aldose reductase. temperature-sensitive mutant of p53 (val135). These S-adenosyl homocystein hydrolase (SAHH) was cells grew actively at 39 1C but arrested at 32 1C, when identified previously in large-scale loss-of-function the p53 protein adopts the active conformation (Car- screen for genes involved in p53-dependent arrest and nero et al., 2000). The antisense fragments inhibited the their role in cancer has been published previously in cell cycle arrest dependent on p53 with rates ranging Berns et al.(2004). Suppression of SAHH by ShRNA between 20 and 40% (Figure 2a). confers resistance to both p53-dependent and p19ARF- dependent proliferation arrest; it also abolishes a DNA- The antisenses affect the transcriptional activity of p53 damage-induced G1 cell-cycle arrest (Berns et al., 2004) We examined the effect of the antisense fragments and is significantly downregulated in colon and lung on the p53-dependent transcriptional activation.