Escape from premature senescence is not sufficient for oncogenic transformation by Ras

Resistance of primary cells to transformation by oncogenic Ras has been attributed to the induction of replicative growth arrest. This irreversible 'fail-safe mechanism' resembles senescence and requires induction by Ras of p19ARF and p53 (refs 3-5). Mutation of either p19ARF or p53 alleviates Ras-induced senescence and facilitates oncogenic transformation by Ras. Here we report that, whereas Rb and p107 are each dispensable for Ras-induced replicative arrest, simultaneous ablation of both genes disrupts Ras-induced senescence and results in unrestrained proliferation. This occurs despite activation by Ras of the p19ARF /p53 pathway, identifying pRb and p107 as essential mediators of Ras-induced antiproliferative p19ARF/p53 signalling. Unexpectedly, in contrast to p19ARF or p53 deficiency, loss of Rb/p107 function does not result in oncogenic transformation by Ras, as Ras-expressing Rb-/-/p107-/- fibroblasts fail to grow anchorage-independently in vitro and are not tumorigenic in vivo. These results demonstrate that in the absence of both Rb and p107 cells are resistant to p19ARF/p53-dependent protection against Ras-induced proliferation, and uncouple escape from Ras-induced premature senescence from oncogenic transformation.     

Oncogenic ras and p53 cooperate to induce cellular senescence

Oncogenic activation of the mitogen-activated protein (MAP) kinase cascade in murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the ARF/p53 tumor suppressor pathway. To investigate whether p53 is sufficient to induce senescence, we introduced a conditional murine p53 allele (p53(val135)) into p53-null mouse embryonic fibroblasts and examined cell proliferation and senescence in cells expressing p53, oncogenic Ras, or both gene products. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras or activated mek1 with p53 enhanced both p53 levels and activity relative to that observed for p53 alone and produced an irreversible cell cycle arrest that displayed features of cellular senescence. p19(ARF) was required for this effect, since p53(-/-) ARF(-/-) double-null cells were unable to undergo senescence following coexpression of oncogenic Ras and p53. Although the levels of exogenous p53 achieved in ARF-null cells were relatively low, the stabilizing effects of p19(ARF) on p53 could not explain the cooperation between oncogenic Ras and p53 in promoting senescence. Hence, enforced p53 expression without oncogenic ras in p53(-/-) mdm2(-/-) double-null cells produced extremely high p53 levels but did not induce senescence. Taken together, our results indicate that oncogenic activation of the MAP kinase pathway in murine fibroblasts converts p53 into a senescence inducer through both quantitative and qualitative mechanisms.     

Inhibition of p21-mediated ROS accumulation can rescue p21-induced senescence

The cyclin-dependent kinase (CDK) inhibitor p21(Waf1/Cip1/Sdi1) was identified initially as a gene induced in senescent cells and itself has been shown to cause permanent growth arrest/senescence. Reactive oxygen species (ROS), a byproduct of oxidative processes, can also induce an irreversible growth arrest similar to senescence. Here we show that p21 increased intracellular levels of ROS both in normal fibroblasts and in p53-negative cancer cells. N-acetyl-L-cysteine, an ROS inhibitor, rescued p21-induced senescence, showing that ROS elevation is necessary for induction of the permanent growth arrest phenotype. p16(Ink4a), a CDK4- and CDK6-specific inhibitor, failed to increase ROS levels, and cell cycle arrest induced by p16 was reversible following its down-regulation, demonstrating the specificity of this p21 effect. A p21 mutant that lacked the ability to bind proliferating cell nuclear antigen (PCNA) retained the ability to induce both ROS and permanent growth arrest. All of these findings establish that p21 mediates senescence by a mechanism involving ROS accumulation which does not require either its PCNA binding or the CDK inhibitory functions shared with p16.     

Cooperation between p53 and p130(Rb2) in induction of cellular senescence

To determine pathways cooperating with p53 in cellular senescence when the retinoblastoma protein (pRb)/p16INK4a pathway is defunct, we stably transfected the p16INK4a-negative C6 rat glioma cell line with a temperature-sensitive mutant p53. Activation of p53(Val-135) induces a switch in pocket protein expression from pRb and p107 to p130(Rb2) and stalls the cells in late G1, early S-phase at high levels of cyclin E. Maintenance of the arrest depends on the functions of p130(Rb2) repressing cyclin A. Inactivation of p53 in senescent cultures restores the pocket proteins to initial levels and initiates progression into S-phase, but the cells fail to resume proliferation, likely due to DNA damage becoming apparent in the arrest and activating apoptosis subsequent to the release from p53-dependent growth suppression. The data indicate that p53 can cooperate selectively with p130(Rb2) to induce cellular senescence, a pathway that may be relevant when the pRb/p16INK4a pathway is defunct.     

p16INK4a-induced senescence is disabled by melanoma-associated mutations

The p16(INK4a)-Rb tumour suppressor pathway is required for the initiation and maintenance of cellular senescence, a state of permanent growth arrest that acts as a natural barrier against cancer progression. Senescence can be overcome if the pathway is not fully engaged, and this may occur when p16(INK4a) is inactivated. p16(INK4a) is frequently altered in human cancer and germline mutations affecting p16(INK4a) have been linked to melanoma susceptibility. To characterize the functions of melanoma-associated p16(INK4a) mutations, in terms of promoting proliferative arrest and initiating senescence, we utilized an inducible expression system in a melanoma cell model. We show that wild-type p16(INK4a) promotes rapid cell cycle arrest that leads to a senescence programme characterized by the appearance of chromatin foci, activation of acidic beta-galactosidase activity, p53 independence and Rb dependence. Accumulation of wild-type p16(INK4a) also promoted cell enlargement and extensive vacuolization independent of Rb status. In contrast, the highly penetrant p16(INK4a) variants, R24P and A36P failed to arrest cell proliferation and did not initiate senescence. We also show that overexpression of CDK4, or its homologue CDK6, but not the downstream kinase, CDK2, inhibited the ability of wild-type p16(INK4a) to promote cell cycle arrest and senescence. Our data provide the first evidence that p16(INK4a) can initiate a CDK4/6-dependent autonomous senescence programme that is disabled by inherited melanoma-associated mutations.     

Cytoplasmic innate immune sensing by the caspase-4 non-canonical inflammasome promotes cellular senescence

Cytoplasmic recognition of microbial lipopolysaccharides (LPS) in human cells is elicited by the caspase-4 and caspase-5 noncanonical inflammasomes, which induce a form of inflammatory cell death termed pyroptosis. Here we show that LPS-mediated activation of caspase-4 also induces a stress response promoting cellular senescence, which is dependent on the caspase-4 substrate gasdermin-D and the tumor suppressor p53. Furthermore, we found that the caspase-4 noncanonical inflammasome is induced and assembled in response to oncogenic RAS signaling during oncogene-induced senescence (OIS). Moreover, targeting caspase-4 expression in OIS showed its critical role in the senescence-associated secretory phenotype and the cell cycle arrest induced in cellular senescence. Finally, we observed that caspase-4 induction occurs in vivo in mouse models of tumor suppression and ageing. Altogether, we are showing that cellular senescence is induced by cytoplasmic LPS recognition by the noncanonical inflammasome and that this pathway is conserved in the cellular response to oncogenic stress.Subject terms: Cell biology, Proteolysis, Immune cell death     

Cellular senescence induced by p53-ras cooperation is independent of p21waf1 in murine embryo fibroblasts

Oncogenic activation in primary murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the p53 tumor suppressor pathway. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras with p53 produced an irreversible cell cycle arrest that displayed features of cellular senescence. Introduction of a conditional murine p53 allele (p53val135) into double p53/p21-null mouse embryonic fibroblasts showed that p21waf1 was not required for this effect, since p53-/-;p21-/- double-null cells undergo terminal growth arrest with features of senescence following coexpression of oncogenic Ras and p53. Our results indicate that oncogenic activation of the Ras pathway in murine fibroblasts converts p53 into a senescence inducer through a p21waf1-independent mechanism.     

Myc,Cdk2 and cellular senescence: Old players,new game

The aberrant activation of oncogenic pathways promotes tumor progression, but concomitantly elicits compensatory tumor-suppressive responses, such as apoptosis or senescence. For example, Ras induces senescence, while Myc generally triggers apoptosis. Myc is in fact viewed as an anti-senescence oncogene, as it is a potent inducer of cell proliferation and immortalization, bypasses growth-inhibitory signals, and cooperates with Ras in cellular transformation. Recent reports prompt re-evaluation of Myc-induced senescence, and of its role in tumor progression and therapy. We have shown that the cyclin-dependent kinase Cdk2, although redundant for cell cycle progression, has a unique role in suppressing a Myc-induced senescence program: Myc activation elicited expression of p16^INK4a and p21^Cip1, and caused senescence in cell lacking Cdk2, but not in Cdk2-proficient cells. Additional cellular activities have been identified that suppress Myc-induced senescence, including the Wrn helicase, Telomerase and Miz1. These senescence-suppressing activities were critical for tumor progression, as deficiency in Cdk2, telomerase or Miz1 reduced the onset of Myc-induced lymphoma in transgenic mice. Other gene products like p53, SUV39H1 or TGFß promoted senescence, which together with apoptosis contributed to tumor suppression. Paradoxically, Myc directly counteracted the very same senescence program that it potentially elicits, since it positively regulated Wrn, Telomerase and Cdk2 activity, and Cdk2 inhibition re-activated the latent senescence program in Myc expressing cells. Hence, while these molecules are instrumental to the oncogenic action of Myc, they may simultaneously constitute its Achille's heel for therapeutic development.     

Contribution of p16INK4a and p21CIP1 pathways to induction of premature senescence of human endothelial cells: permissive role of p53

We have previously found that nonenzymatically glycated collagen I (GC), mimicking diabetic microenvironment, can induce senescent phenotype in early passage human umbilical vein endothelial cells (HUVECs). In the present study, we explored the functional involvement of cell cycle checkpoint pathways in initiating GC-induced premature endothelial cell senescence. When compared with native collagen, early passage HUVECs showed increased p53, p21(CIP1) (p21), and p16(INK4a) (p16) mRNA expression after exposure to GC. Twenty-four hours after transfection of p16, p21, and p53-enhanced green fluorescent protein (EGFP) recombinant plasmids, HUVECs entered G(1)-phase cell cycle arrest. By days 3 and 5, HUVECs transfected with p16-EGFP showed an increased proportion of senescent cells, and this increase was more prominent in the GFP-positive cell population, which exhibited 68% of senescent cells. Transfection of p21 also induced senescence but only by day 5. Cotransfection of p16 and p21 showed no additive effect. Transfection of p21 or p53 induced apoptosis in HUVECs. Next, we suppressed endogenous p53, p21, p16, or retinoblastoma (Rb) gene expression through small interference RNA strategy and investigated their influence in p16- and p21-initiated endothelial cell senescence. Analysis indicated that suppression of p53 expression can abolish senescence induced by p16 overexpression. Paradoxically, this effect was not observed when p21 was suppressed. On the other hand, suppression of Rb eliminated senescence initiated by either p16 or p21 overexpression. In summary, the p53/p21 pathway is mainly responsible for GC-induced apoptosis, but the coordinated activation of the p53/p21 and p16 pathway is responsible for GC-induced endothelial cell senescence through a Rb-dependent mechanism.     

E2F1 and E2F3 activate ATM through distinct mechanisms to promote E1A-induced apoptosis

Deregulation of the Rb-E2F pathway occurs in many cancers and results in aberrant cell proliferation as well as an increased propensity to undergo apoptosis. In most cases, apoptosis in response to Rb inactivation involves the activation of p53 but the molecular details of the signaling pathway connecting Rb loss to p53 are poorly understood. Here we demonstrate that the E1A oncoprotein, which binds and inhibits Rb family members, induces the accumulation and phosphorylation of p53 through the DNA damage-responsive ATM kinase. As a result, E1A-induced apoptosis is significantly impaired in cells lacking ATM. In contrast, inactivation of ARF, which is widely believed to activate p53 in response to oncogenic stress, has no effect on p53 induction and only a modest effect on apoptosis in response to E1A. Both E2F1 and E2F3 contribute to ATM-dependent phosphorylation of p53 and apoptosis in cells expressing E1A. However, deregulated E2F3 activity is implicated in the DNA damage caused by E1A while E2F1 stimulates ATM- and NBS1-dependent p53 phosphorylation and apoptosis through a mechanism that does not involve DNA damage.     

Distinct roles for p107 and p130 in Rb-independent cellular senescence

Telomere attrition, DNA damage and constitutive mitogenic signaling can all trigger cellular senescence in normal cells and serve as a defense against tumor progression. Cancer cells may circumvent this cellular defense by acquiring genetic mutations in checkpoint proteins responsible for regulating permanent cell cycle arrest. A small family of tumor suppressor genes encoding the retinoblastoma susceptibility protein family (Rb, p107, p130) exerts a partially redundant control of entry into S phase of DNA replication and cellular proliferation. Here we report that activation of the p53-dependent DNA damage response has been found to accelerate senescence in human prostate cancer cells lacking a functional Rb protein. This novel form of irradiation-induced premature cellular senescence reinforces the notion that other Rb family members may compensate for loss of Rb protein in the DNA damage response pathway. Consistent with this hypothesis, depletion of p107 potently inhibits the irradiation-induced senescence observed in DU145 cells. In contrast, p130 depletion triggers a robust and unexpected form of premature senescence in unirradiated cells. The dominant effect of depleting both p107 and p130, in the absence of Rb, was a complete blockade of irradiation-induced cellular senescence. Onset of the p107-dependent senescence was temporally associated with p53-mediated stabilization of the cyclin-dependent kinase inhibitor p27 and decreases in c-myc and cks1 expression. These results indicate that p107 is required for initiation of accelerated cellular senescence in the absence of Rb and introduces the concept that p130 may be required to prevent the onset of terminal growth arrest in unstimulated prostate cancer cells lacking a functional Rb allele.     

细胞衰老与肿瘤发生

细胞衰老（cell senescence）是指细胞在信号转导作用下不可逆地脱离细胞周期并丧失增殖能力后进入的一种相对稳定的状态。细胞衰老有增殖衰老与早熟衰老两种形式：增殖衰老由端粒缩短激发的信号转导激发,与TP53/CDKN1a（p21^WAF-1/Cip1）/pRB/E2F信号通路密切相关;早熟衰老由细胞内在或外在急慢性应激信号引发,与TP53/CDKN1a（p21^WAF-1/Cip1）/pRB/E2F或CDKN2a（p16^ink4A）/pRB/E2F信号通路相关。目前研究已经证实早熟衰老是细胞在癌变过程中的天然屏障,是继DNA修复、细胞凋亡后的第三大细胞内在抗癌机制,在机体防止肿瘤形成中起重要作用。     

Repression of the human papillomavirus E6 gene initiates p53-dependent, telomerase-independent senescence and apoptosis in HeLa cervical carcinoma cells

Cervical cancer cells express high-risk human papillomavirus (HPV) E6 and E7 proteins. When both HPV oncogenes are repressed in HeLa cervical carcinoma cells, the dormant p53 and retinoblastoma (Rb) tumor suppressor pathways are activated, and the cells undergo senescence in the absence of apoptosis. When the E6 gene is repressed in cells that continue to express an E7 gene, the p53 pathway, but not the Rb pathway, is activated, and both senescence and apoptosis are triggered. To determine the role of p53 signaling in senescence or apoptosis after repression of HPV oncogenes, we introduced a dominant-negative allele of p53 into HeLa cells. Dominant-negative p53 prevented senescence and apoptosis when E6 alone was repressed but did not inhibit senescence when both E6 and E7 were repressed. To determine whether reduced telomerase activity was involved in senescence or apoptosis after E6 repression, we generated HeLa cells stably expressing an exogenous hTERT gene, which encodes the catalytic subunit of telomerase. Although these cells contained markedly elevated telomerase activity and elongated telomeres, hTERT expression did not prevent senescence and apoptosis when E6 alone was repressed. These results demonstrate that when the Rb tumor suppressor pathway is inactivated by the E7 protein, E6 repression activates p53 signaling, which in turn is required for growth inhibition, senescence, and apoptosis. Thus, sustained inactivation of the p53 pathway by the E6 protein is required for maintenance of the proliferative phenotype of HeLa cervical carcinoma cells.