A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy

p53 and INK4a/ARF mutations promote tumorigenesis and drug resistance, in part, by disabling apoptosis. We show that primary murine lymphomas also respond to chemotherapy by engaging a senescence program controlled by p53 and p16(INK4a). Hence, tumors with p53 or INK4a/ARF mutations-but not those lacking ARF alone-respond poorly to cyclophosphamide therapy in vivo. Moreover, tumors harboring a Bcl2-mediated apoptotic block undergo a drug-induced cytostasis involving the accumulation of p53, p16(INK4a), and senescence markers, and typically acquire p53 or INK4a mutations upon progression to a terminal stage. Finally, mice bearing tumors capable of drug-induced senescence have a much better prognosis following chemotherapy than those harboring tumors with senescence defects. Therefore, cellular senescence contributes to treatment outcome in vivo.     

INK4a/ARF limits the expansion of cells suffering from replication stress

Replication stress (RS) is a source of DNA damage that has been linked to cancer and aging, which is suppressed by the ATR kinase. In mice, reduced ATR levels in a model of the ATR-Seckel syndrome lead to RS and accelerated aging. Similarly, ATR-Seckel embryonic fibroblasts (MEF) accumulate RS and undergo cellular senescence. We previously showed that senescence of ATR-Seckel MEF cannot be rescued by p53-deletion. Here, we show that the genetic ablation of the INK4a/Arf locus fully rescues senescence on ATR mutant MEF, but also that induced by other conditions that generate RS such as low doses of hydroxyurea or ATR inhibitors. In addition, we show that a persistent exposure to RS leads to increased levels of INK4a/Arf products, revealing that INK4a/ARF behaves as a bona fide RS checkpoint. Our data reveal an unknown role for INK4a/ARF in limiting the expansion of cells suffering from persistent replication stress, linking this well-known tumor suppressor to the maintenance of genomic integrity.     

Ser18 and Ser23 Phosphorylation Plays Synergistic Roles in Activating p53-Dependent Neuronal Apoptosis

Tumor suppressor p53 is required for the neuronal apoptosis in response to DNA double-stranded break (DSB) damage. Posttranslational modifications such as phosphorylation play important roles in activating p53-dependent apoptosis after DNA damage. In support of this notion, our recent studies indicate that Ser18 and Ser23 phosphorylation together plays critical roles in activating p53 apoptotic activities in vivo. Thymocytes derived from p53S18/23A mice are essentially resistant to p53-dependent apoptosis after DNA DSB damage. In addition, identical to p53-deficiency, p53S18/23A knock-in mutation completely rescues the embryonic lethality of XRCC4-/- mice, which die of the massive p53-dependent apoptosis of embryonic neurons likely as a result of accumulated endogenous DNA damage. To dissect the contribution of Ser18 and Ser23 phosphorylation to p53-dependent neuronal apoptosis, we report here that neither p53S18A nor p53S23A mutation alone can rescue the embryonic lethality of XRCC4-/- mice. Therefore, Ser18 and Ser23 phosphorylation plays synergistic and critical roles in activating p53-dependent neuronal apoptosis.     

Ser18 and 23 phosphorylation is required for p53-dependent apoptosis and tumor suppression

Mouse p53 is phosphorylated at Ser18 and Ser23 after DNA damage. To determine whether these two phosphorylation events have synergistic functions in activating p53 responses, we simultaneously introduced Ser18/23 to Ala mutations into the endogenous p53 locus in mice. While partial defects in apoptosis are observed in p53S18A and p53S23A thymocytes exposed to IR, p53-dependent apoptosis is essentially abolished in p53S18/23A thymocytes, indicating that these two events have critical and synergistic roles in activating p53-dependent apoptosis. In addition, p53S18/23A, but not p53S18A, could completely rescue embryonic lethality of Xrcc4(-/-) mice that is caused by massive p53-dependent neuronal apoptosis. However, certain p53-dependent functions, including G1/S checkpoint and cellular senescence, are partially retained in p53(S18/23A) cells. While p53(S18A) mice are not cancer prone, p53S18/23A mice developed a spectrum of malignancies distinct from p53S23A and p53(-/-) mice. Interestingly, Xrcc4(-/-)p53S18/23A mice fail to develop tumors like the pro-B cell lymphomas uniformly developed in Xrcc4(-/-) p53(-/-) animals, but exhibit developmental defects typical of accelerated ageing. Therefore, Ser18 and Ser23 phosphorylation is important for p53-dependent suppression of tumorigenesis in certain physiological context.     

An essential role for REV3 in mammalian cell survival: absence of REV3 induces p53-independent embryonic death

The REV3 gene of budding yeast encodes the catalytic subunit of DNA polymerase zeta that carries out translesion DNA synthesis. While REV3-null yeast mutants are viable and exhibit normal growth, Rev3-deficient mice die around midgestation of embryogenesis, which is accompanied by massive apoptosis of cells within the embryo proper. We have investigated whether REV3 is required for the survival of mouse cells and whether the embryonic lethality caused by REV3 deficiency can be rescued by introduction of a Rev3 transgene or by inactivation of p53, the cellular gatekeeper that regulates DNA damage-induced apoptosis. We show that Rev3(-/-) blastocysts were unable to survive and grow in culture but expression of a Rev3 transgene restored their outgrowth. Moreover, Rev3 transgene expression suppressed the apoptosis in E7.5 Rev3(-/-) embryos. The Rev3(-/-) embryonic lethality, however, was not rescued by either Rev3 transgene expression or p53 deficiency. These results reveal an essential role for REV3 in the survival and growth of mammalian cells and suggest that Rev3(-/-) embryonic death occurs in a p53-independent pathway.     

Loss of Borealin/DasraB leads to defective cell proliferation, p53 accumulation and early embryonic lethality

Borealin/DasraB is a member of the chromosomal passenger protein complex (CPC) required for proper segregation of chromosomes during mitosis. In Drosophila melanogaster, inactivation of Borealin/DasraB results in polyploidy, delayed mitosis and abnormal tissue development, indicating its critical role for cell proliferation. However, the in vivo role of mammalian Borealin/DasraB remains unclear. Here, we analyzed the expression of Borealin/DasraB and found that borealin is widely expressed in embryonic tissues and later restricted to adult tissues which relies on rapid cell proliferation. To determine the role of borealin during mouse development, we generated borealin-null mice through targeted disruption. While heterozygous mice developed normally, disruption of both borealin alleles resulted in early embryonic lethality by 5.5 dpc (days postcoitus) due to mitotic defects and apoptosis in blastocyst cells that showed microtubule disorganization and no CPC enrichment. At 5.5 dpc, borealin-null embryos exhibited excessive apoptosis and elevated expression of p53. However, loss of p53 did not abrogate or delay embryonic lethality, revealing that Borealin/DasraB inactivation triggered impaired mitosis and apoptosis though p53-independent mechanisms. Our data show that Borealin/DasraB is essential for cell proliferation during early embryonic development, and its early embryonic lethality cannot be rescued by the loss of p53.     

Replicative senescence as a barrier to human cancer

There is evidence that one critically short telomere may be recognized as DNA damage and, as a consequence, induce a p53/p21WAF- and p16INK4A-dependent G1 cell cycle checkpoint to cause senescence. Additionally, senescence via a p53- and p16(INK4A)-dependent mechanism can be induced by the over- or under-stimulation of certain signalling pathways that are involved in cancer. Central to this alternative senescence mechanism is the p14ARF protein, which connects oncogene activation, but not DNA damage, to p53 activation and senescence. We find that immortal keratinocytes almost invariably have dysfunctional p53 and p16 and have high levels of telomerase, but very often express a wild-type p14(ARF). Furthermore, when normal keratinocytes senesce they show a striking elevation of p16 protein, but not of p14(ARF) or its downstream targets p53 and p21(WAF). These results suggest that p16, rather than p14(ARF), is the more important gene in human keratinocyte senescence, but do not exclude a co-operative role for p14(ARF), perhaps in the induction of senescence by activated oncogenes in neoplasia. Regardless of mechanism, these results suggest that replicative senescence acts as a barrier to human cancer development.     

DNA damage induces MDMX nuclear translocation by p53-dependent and -independent mechanisms

The MDM2 homolog MDMX is an important regulator of p53 activity during embryonic development. MDMX inactivation in mice results in embryonic lethality in a p53-dependent fashion. The expression level of MDMX is not induced by DNA damage, and its role in stress response is unclear. We show here that ectopically expressed MDMX is mainly localized in the cytoplasm. DNA damage promotes nuclear translocation of MDMX in cells with or without p53. Coexpression of MDM2 or p53 is sufficient to induce MDMX nuclear translocation, suggesting that activation of p53 and induction of MDM2 expression can contribute to this process. Stable transfection of MDMX into U2OS cells does not alter p53 level but results in reduced p53 DNA-binding activity and reduced MDM2 expression. The ability of ARF (alternate reading frame of INK4a) to activate p53 is also significantly inhibited by expression of MDMX. These results suggest that MDMX function may be regulated by DNA damage. Furthermore, MDMX may complement MDM2 in regulating p53 during embryonic development due to its ability to inhibit p53 in the presence of ARF.     

ATM-Chk2-p53 activation prevents tumorigenesis at an expense of organ homeostasis upon Brca1 deficiency

BRCA1 is a checkpoint and DNA damage repair gene that secures genome integrity. We have previously shown that mice lacking full-length Brca1 (Brca1(delta11/delta11)) die during embryonic development. Haploid loss of p53 completely rescues embryonic lethality, and adult Brca1(delta11/delta11)p53+/- mice display cancer susceptibility and premature aging. Here, we show that reduced expression and/or the absence of Chk2 allow Brca1(delta11/delta11) mice to escape from embryonic lethality. Compared to Brca1(delta11/delta11)p53+/- mice, lifespan of Brca1(delta11/delta11)Chk2-/- mice was remarkably extended. Analysis of Brca1(delta11/delta11)Chk2-/- mice revealed that p53-dependent apoptosis and growth defect caused by Brca1 deficiency are significantly attenuated in rapidly proliferating organs. However, in later life, Brca1(delta11/delta11)Chk2-/- female mice developed multiple tumors. Furthermore, haploid loss of ATM also rescued Brca1 deficiency-associated embryonic lethality and premature aging. Thus, in response to Brca1 deficiency, the activation of the ATM-Chk2-p53 signaling pathway contributes to the suppression of neoplastic transformation, while leading to compromised organismal homeostasis. Our data highlight how accurate maintenance of genomic integrity is critical for the suppression of both aging and malignancy, and provide a further link between aging and cancer.     

The molecular scaffold kinase suppressor of Ras 1 is a modifier of RasV12-induced and replicative senescence

In primary mouse embryo fibroblasts (MEFs), oncogenic Ras induces growth arrest via Raf/MEK/extracellular signal-regulated kinase (ERK)-mediated activation of the p19ARF/p53 and INK4/Rb tumor suppressor pathways. Ablation of these same pathways causes spontaneous immortalization in MEFs, and oncogenic transformation by Ras requires ablation of one or both of these pathways. We show that Kinase Suppressor of Ras 1 (KSR1), a molecular scaffold for the Raf/MEK/ERK cascade, is necessary for RasV12-induced senescence, and its disruption enhances primary MEF immortalization. RasV12 failed to induce p53, p19ARF, p16INK4a, and p15INK4b expression in KSR1-/- MEFs and increased proliferation instead of causing growth arrest. Reintroduction of wild-type KSR1, but not a mutated KSR1 construct unable to bind activated ERK, rescued RasV12-induced senescence. On continuous culture, deletion of KSR1 accelerated the establishment of spontaneously immortalized cultures and increased the proportion of cultures escaping replicative crisis. Despite enhancing escape from both RasV12-induced and replicative senescence, however, both primary and immortalized KSR1-/- MEFs are completely resistant to RasV12-induced transformation. These data show that escape from senescence is not necessarily a precursor for oncogenic transformation. Furthermore, these data indicate that KSR1 is a member of a unique class of proteins whose deletion blocks both senescence and transformation.     

INK4a deletion results in improved kidney regeneration and decreased capillary rarefaction after ischemia-reperfusion injury

The molecular mechanisms that lead to tubular atrophy, capillary loss, and fibrosis following acute kidney injury are not very clear but may involve cell cycle inhibition by increased expression of cyclin kinase inhibitors. The INK4a/ARF locus encodes overlapping genes for two proteins, a cyclin kinase inhibitor, p16(INK4a), and a p53 stabilizer, p19(ARF), from independent promoters. To determine if decreased INK4a gene expression results in improved kidney regeneration, INK4a knockout (KO) and wild-type (WT) mice were subjected to ischemia-reperfusion injury (IRI). p16(INK4a) and p19(ARF) levels were increased markedly in WT mice at 1-28 days after injury. Kidneys were examined to determine the localization and levels of p16(INK4a), apoptosis, cell proliferation, and capillary rarefaction. KO mice displayed decreased tubular cell apoptosis, increased cell proliferation, and lower creatinine levels after injury. KO mice had significantly higher capillary density compared with WT mice at 14-42 days after IRI. Plasma granulocyte colony-stimulating factor (G-CSF) increased after ischemia in both WT and KO mice and was elevated markedly in KO compared with WT mice. KO kidney digests contained higher counts of Gr-1(+)/Cd11b(+) myeloid cells by flow cytometry. KO mice treated with a Gr-1-depleting antibody displayed reduced vascular endothelial growth factor mRNA, plasma G-CSF, and capillary density, and an increase in serum creatinine and medullary myofibroblasts, compared with untreated KO mice 14 days after ischemia. The anti-angiogenic effect of Gr-1 depletion in KO mice was confirmed by Matrigel angiogenesis assays. These results suggest that the absence of p16(INK4a) and p19(ARF) following IRI has a protective effect on the kidney through improved epithelial and microvascular repair, in part by enhancing the mobilization of myeloid cells into the kidney.     

Additive effect of TAp63 deficiency on the adrenocortical dysplasia (acd) phenotype

Adrenocortical dysplasia (acd) is a spontaneous autosomal recessive mouse mutation exhibiting caudal truncation, vertebral segmentation defects, hydronephrosis, limb hypoplasia, and perinatal lethality. Acd encodes TPP1, a component of the shelterin complex that maintains telomere integrity, and consequently acd mutant mice have telomere dysfunction and genomic instability. We previously showed that apoptosis is the primary mechanism causing the acd skeletal phenotype, and that p53 deficiency rescues the skeletal defects of the acd phenotype but has no effect on the perinatal lethality. The Trp63 gene encodes multiple isoforms, which play a role in proliferation, apoptosis, and stem/progenitor cell maintenance. Different p63 isoforms exhibit both proapoptotic (TAp63) and antiapoptotic (ΔNp63) functions. We hypothesized that deficiency of proapoptotic TAp63 isoforms might rescue the acd skeletal phenotype, similar to our previous observations with deficiency of p53. Mice heterozygous for a null allele of TAp63 were crossed to heterozygous acd mice to determine the effect of TAp63 deficiency on the acd mutant phenotype. In contrast to our results with the acd?×?p53 cross, skeletal anomalies were not rescued by deficiency of TAp63. In fact, the limb and vertebral anomalies observed in double-mutant embryos were more severe than those of embryos with the acd mutation alone, demonstrating a dose-dependent effect. These studies suggest that TAp63 isoforms do not facilitate p53-like apoptosis during development in response to acd-mediated telomere dysfunction and are consistent with the proposed roles of TAp63 in maintaining genomic stability.     

p53 Deficiency rescues neuronal apoptosis but not differentiation in DNA polymerase beta-deficient mice

In mammalian cells, DNA polymerase beta (Polbeta) functions in base excision repair. We have previously shown that Polbeta-deficient mice exhibit extensive neuronal cell death (apoptosis) in the developing nervous system and that the mice die immediately after birth. Here, we studied potential roles in the phenotype for p53, which has been implicated in DNA damage sensing, cell cycle arrest, and apoptosis. We generated Polbeta(-/-) p53(-/-) double-mutant mice and found that p53 deficiency dramatically rescued neuronal apoptosis associated with Polbeta deficiency, indicating that p53 mediates the apoptotic process in the nervous system. Importantly, proliferation and early differentiation of neuronal progenitors in Polbeta(-/-) p53(-/-) mice appeared normal, but their brains obviously displayed cytoarchitectural abnormalities; moreover, the mice, like Polbeta(-/-) p53(+/+) mice, failed to survive after birth. Thus, we strongly suggest a crucial role for Polbeta in the differentiation of specific neuronal cell types.     

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.     

Cellular senescence induced by cathepsin X downregulation

Cellular senescence represents a powerful tumor suppressor mechanism to prevent proliferation and invasion of malignant cells. Since tumor cells as well as primary fibroblasts lacking the lysosomal cysteine-type carboxypeptidase cathepsin X exhibit a reduced invasive capacity, we hypothesized that the underlying reason may be the induction of cellular senescence. To investigate the cellular and molecular mechanisms leading to diminished migration/invasion of cathepsin X-deficient cells, we have analyzed murine embryonic fibroblasts (MEF) derived from cathepsin X-deficient mice and neonatal human dermal fibroblasts (NHDF) transfected with siRNAs targeting cathepsin X. Remarkably, both cell types exhibited a flattened and enlarged cell body, a characteristic phenotype of senescent cells. Additional evidence for accelerated senescence was obtained by detection of the common senescence marker β-galactosidase. Further examination revealed increased expression levels of senescence-associated genes such as p16, p21, p53, and caveolin in these cells along with a reduced proliferation rate. The accelerated cellular senescence induced by cathepsin X deficiency was rescued by simultaneous expression of exogenous cathepsin X. Finally, cell cycle analysis confirmed a marked reduction of the synthesis rate and prolongation of the S-phase, while susceptibility to apoptosis of cathepsin X-deficient cells remained unchanged. In conclusion, cathepsin X deficiency leads to accelerated cellular senescence and consequently to diminished cellular proliferation and migration/invasion implying a potential role of cathepsin X in bypassing cellular senescence.