Mutant p53 tumor suppressor alleles release ras-induced cell cycle growth arrest.

Overexpression of an activated ras gene in the rat embryo fibroblast line REF52 results in growth arrest at either the G1/S or G2/M boundary of the cell cycle. Both the DNA tumor virus proteins simian virus 40 large T antigen and adenovirus 5 E1a are able to rescue ras induced lethality and cooperate with ras to fully transform REF52 cells. In this report, we present evidence that the wild-type activity of the tumor suppressor gene p53 is involved in the negative growth regulation of this model system. p53 genes encoding either a p53Val-135 or p53Pro-193 mutation express a highly stable p53 protein with a conformation-dependent loss of wild-type activity and the ability to eliminate any endogenous wild-type p53 activity in a dominant negative manner. In cotransfection assays, these mutant p53 genes are able to rescue REF52 cells from ras-induced growth arrest, resulting in established cell lines which express elevated levels of the ras oncoprotein and show morphological transformation. Full transformation, as assayed by tumor formation in nude mice, is found only in the p53Pro-193-plus-ras transfectants. These cells express higher levels of the ras protein than do the p53Val-135-plus-ras-transfected cells. Transfection of REF52 cells with ras alone or a full-length genomic wild-type p53 plus ras results in growth arrest and lethality. Therefore, the selective event for p53 inactivation or loss during tumor progression may be to overcome a cell cycle restriction induced by oncogene overexpression (ras). These results suggest that a normal function of p53 may be to mediate negative growth regulation in response to ras or other proliferative inducing signals.     

Ing1 mediates p53 accumulation and chromatin modification in response to oncogenic stress

ING proteins are putative tumor suppressor proteins linked to the p53 pathway and to the chromatin modification machinery. Here we have analyzed the role of the products of the murine Ing1 locus in cellular tumor-protective responses, using mouse primary fibroblasts where the Ing1 locus has been inactivated by the integration of a betageo cassette. We show that Ing1-deficient mouse embryonic fibroblasts display a defective senescence-like antiproliferative response against oncogenic Ras, affecting several senescence-specific markers. This phenotype is accompanied by a reduced accumulation of p53, which can be explained by the reduced basal p53 protein stability in the Ing1-deficient background. Ing1 deficiency also results in defects in the appearance of heterochromatic marks upon expression of oncogenic Ras, suggestive of impaired heterochromatin formation during oncogene-induced senescence. Our results support an important role for the Ing1 locus in protection against oncogenic stress in vivo, both as a mediator of p53 activation and as a regulator of chromatin remodeling processes.     

PIDDosome-independent tumor suppression by Caspase-2

The PIDDosome, a multiprotein complex constituted of the 'p53-induced protein with a death domain (PIDD), 'receptor-interacting protein (RIP)-associated ICH-1/CED-3 homologous protein with a death domain' (RAIDD) and pro-Caspase-2 has been defined as an activating platform for this apoptosis-related protease. PIDD has been implicated in p53-mediated cell death in response to DNA damage but also in DNA repair and nuclear factor kappa-light-chain enhancer (NF-κB) activation upon genotoxic stress, together with RIP-1 kinase and Nemo/IKKγ. As all these cellular responses are critical for tumor suppression and deregulated expression of individual PIDDosome components has been noted in human cancer, we investigated their role in oncogenesis induced by DNA damage or oncogenic stress in gene-ablated mice. We observed that Pidd or Caspase-2 failed to suppress lymphoma formation triggered by γ-irradiation or 3-methylcholanthrene-driven fibrosarcoma development. In contrast, Caspase-2 showed tumor suppressive capacity in response to aberrant c-Myc expression, which did not rely on PIDD, the BH3-only protein Bid (BH3 interacting domain death agonist) or the death receptor ligand Trail (TNF-related apoptosis-inducing ligand), but associated with reduced rates of p53 loss and increased extranodal dissemination of tumor cells. In contrast, Pidd deficiency associated with abnormal M-phase progression and delayed disease onset, indicating that both proteins are differentially engaged upon oncogenic stress triggered by c-Myc, leading to opposing effects on tumor-free survival.     

53BP1 cooperates with p53 and functions as a haploinsufficient tumor suppressor in mice

p53 binding protein 1 (53BP1) is a putative DNA damage sensor that accumulates at sites of double-strand breaks (DSBs) in a manner dependent on histone H2AX. Here we show that the loss of one or both copies of 53BP1 greatly accelerates lymphomagenesis in a p53-null background, suggesting that 53BP1 and p53 cooperate in tumor suppression. A subset of 53BP1-/- p53-/- lymphomas, like those in H2AX-/- p53-/- mice, were diploid and harbored clonal translocations involving antigen receptor loci, indicating misrepair of DSBs during V(D)J recombination as one cause of oncogenic transformation. Loss of a single 53BP1 allele compromised genomic stability and DSB repair, which could explain the susceptibility of 53BP1+/- mice to tumorigenesis. In addition to structural aberrations, there were high rates of chromosomal missegregation and accumulation of aneuploid cells in 53BP1-/- p53+/+ and 53BP1-/- p53-/- tumors as well as in primary 53BP1-/- splenocytes. We conclude that 53BP1 functions as a dosage-dependent caretaker that promotes genomic stability by a mechanism that preserves chromosome structure and number.     

A role for Xrcc2 in the early stages of mouse development

Xrcc2 is one of a family of five Rad51-like genes with important roles in the repair of DNA damage by homologous recombination (HR) in mammals. We have shown previously that loss of Xrcc2 in mice results in severe but variable developmental defects and embryonic lethality, potentially linked to excessive apoptosis. To look at the causes of lethality, and possibly to allow Xrcc2-/- mice to survive to birth, we have produced double knockout mice deficient in either the p53 oncoprotein or Ataxia telangiectasia mutated (Atm). Overall we show that the excessive apoptosis observed in Xrcc2-/- embryos is p53-dependent, and that loss of p53 can restore growth capacity to Xrcc2-/- fibroblasts in culture, but that it cannot rescue the embryonic lethality. Additionally, although the Xrcc2-/- Trp53-/- embryos show a near-normal morphology they remain relatively small in size. Loss of Atm in an Xrcc2-/- embryo has little effect, suggesting that response to loss of HR capacity is not mediated through the Atm kinase in the early stages of mouse development. Further, as seen by reduced expression of the early developmental marker, Delta-like1, the normal developmental programme is perturbed in Xrcc2-/- embryonic tissues, particularly during neurogenesis and somitogenesis. Taken together our data suggest that the accumulation of spontaneous damage in HR-deficient embryos has severe consequences for the development and survival of mammals due to the unregulated loss of cells important to the developmental programme.     

neu/ERBB2 cooperates with p53-172H during mammary tumorigenesis in transgenic mice.

Thirty percent of human breast cancers have amplification of ERBB2, often in conjunction with mutations in p53. The most common p53 mutation in human breast cancers is an Arg-to-His mutation at codon 175, an allele that functions in a dominant oncogenic manner in tumorigenesis assays and is thus distinct from loss of p53. Transgenic mice expressing mouse mammary tumor virus-driven neu transgene (MMTV-neu) develop clonal mammary tumors with a latency of 234 days, suggesting that other events are necessary for tumor development. We have examined the role of mutations in p53 in tumor development in these mice. We have found that 37% of tumors arising in these mice have a missense mutations in p53. We have directly tested for cooperativity between neu and mutant p53 in mammary tumorigenesis by creating bitransgenic mice carrying MMTV-neu and 172Arg-to-His p53 mutant (p53-172H). In these bitransgenic mice, tumor latency is shortened to 154 days, indicating strong cooperativity. None of the nontransgenic mice or the p53-172H transgenic mice developed tumors within this time period. Tumors arising in the p53-172H/neu bitransgenic mice were anaplastic and aneuploid and exhibited increased apoptosis, in distinction to tumors arising in p53-null mice, in which apoptosis is diminished. Further experiments address potential mechanisms of cooperativity between the two transgenes. In these bitransgenic mice, we have recapitulated two common genetic lesions that occur in human breast cancer and have shown that p53 mutation is an important cooperating event in neu-mediated oncogenesis.     

The ability of large T antigen to complex with p53 is necessary for the increased life span and partial transformation of human cells by simian virus 40.

Simian virus 40 (SV40) T antigen binds to the tumor suppressor p53 protein, and this association may contribute to oncogenic transformation by the virus. We investigated the importance of this binding on transformation by examining three replication-competent mutants of SV40 (402DE, 402DN, and 402DH). These mutants express T antigens defective in binding to human and monkey p53s but retain some binding with mouse p53. All showed significant reduction in their ability to induce transformed cell foci of two normal human cell lines as well as a slight reduction with mouse embryo cells. Other comparable mutants which express T antigens retaining the ability to complex with p53 were able to induce foci at wild-type levels in both human and mouse cells. Further studies were performed with five T-antigen-positive clones isolated from the few human cell foci that appeared after transfection with 402 mutant DNAs. All five clones reached senescence at about the same point as did the parental untransformed cells. However, six other human cell clones obtained after transfection with DNA from nondefective mutants or wild-type virus were still growing well at more than 10 passages beyond their expected life span. These results suggest that the ability of T antigen to form stable complexes with p53 is necessary for SV40 to extend the life span and partially transform human cells in culture.     

Irradiation Selects for p53-Deficient Hematopoietic Progenitors

Identification and characterization of mutations that drive cancer evolution constitute a major focus of cancer research. Consequently, dominant paradigms attribute the tumorigenic effects of carcinogens in general and ionizing radiation in particular to their direct mutagenic action on genetic loci encoding oncogenes and tumor suppressor genes. However, the effects of irradiation are not limited to genetic loci that encode oncogenes and tumor suppressors, as irradiation induces a multitude of other changes both in the cells and their microenvironment which could potentially affect the selective effects of some oncogenic mutations. P53 is a key tumor suppressor, the loss of which can provide resistance to multiple genotoxic stimuli, including irradiation. Given that p53 null animals develop T-cell lymphomas with high penetrance and that irradiation dramatically accelerates lymphoma development in p53 heterozygous mice, we hypothesized that increased selection for p53-deficient cells contributes to the causal link between irradiation and induction of lymphoid malignancies. We sought to determine whether ionizing irradiation selects for p53-deficient hematopoietic progenitors in vivo using mouse models. We found that p53 disruption does not provide a clear selective advantage within an unstressed hematopoietic system or in previously irradiated BM allowed to recover from irradiation. In contrast, upon irradiation p53 disruption confers a dramatic selective advantage, leading to long-term expansion of p53-deficient clones and to increased lymphoma development. Selection for cells with disrupted p53 appears to be attributable to several factors: protection from acute irradiation-induced ablation of progenitor cells, prevention of irradiation-induced loss of clonogenic capacity for stem and progenitor cells, improved long-term maintenance of progenitor cell fitness, and the disabling/elimination of competing p53 wild-type progenitors. These studies indicate that the carcinogenic effect of ionizing irradiation can in part be explained by increased selection for cells with p53 disruption, which protects progenitor cells both from immediate elimination and from long-term reductions in fitness following irradiation.     

Opposing effects of Ras on p53: transcriptional activation of mdm2 and induction of p19ARF

Mdm2 acts as a major regulator of the tumor suppressor p53 by targeting its destruction. Here, we show that the mdm2 gene is also regulated by the Ras-driven Raf/MEK/MAP kinase pathway, in a p53-independent manner. Mdm2 induced by activated Raf degrades p53 in the absence of the Mdm2 inhibitor p19ARF. This regulatory pathway accounts for the observation that cells transformed by oncogenic Ras are more resistant to p53-dependent apoptosis following exposure to DNA damage. Activation of the Ras-induced Raf/MEK/MAP kinase may therefore play a key role in suppressing p53 during tumor development and treatment. In primary cells, Raf also activates the Mdm2 inhibitor p19ARF. Levels of p53 are therefore determined by opposing effects of Raf-induced p19ARF and Mdm2.     

Synthetic lethality between Rb, p53 and Dicer or miR-17-92 in retinal progenitors suppresses retinoblastoma formation

Synthetic lethality is a promising strategy for specific targeting of cancer cells that carry mutations that are absent in normal cells. This approach may help overcome the challenge associated with targeting dysfunctional tumour suppressors, such as p53 and Rb?(refs?, ). Here we show that Dicer1 targeting prevents retinoblastoma formation in mice by synthetic lethality with combined inactivation of p53 and Rb. Although Dicer1 functions as a haploinsufficient tumour suppressor, its complete loss of function is selected against during tumorigenesis. We show that Dicer1 deficiency is tolerated in Rb-deficient retinal progenitor cells harbouring an intact p53 pathway, but not in the absence of p53. This synthetic lethality is mediated by the oncogenic miR-17-92 cluster because its deletion phenocopies Dicer1 loss in this context. miR-17-92?inactivation suppresses retinoblastoma formation in mice and co-silencing of miR-17/20a and p53 cooperatively decreases the viability of human retinoblastoma cells. These data provide an explanation for the selective pressure against loss of Dicer1 during tumorigenesis and a proof-of-concept that targeting miRNAs may potentially represent a general approach for synthetic lethal targeting of cancer cells that harbour specific cancer-inducing genotypes.     

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.     

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.     

An unexpected role for p53 in augmenting SV40 large T antigen-mediated tumorigenesis

Simian virus 40 large T antigen transforms cells by sequestration and inactivation of the tumor suppressor proteins p53, retinoblastoma gene product (pRb), and the pRb-related proteins p107 and p130. Thus, the absence of functional p53 is expected to promote T antigen-mediated tumorigenesis. However, in a transgenic mouse model of T antigen-mediated beta cell carcinogenesis (Rip1Tag2), tumor volumes are significantly diminished when these mice are intercrossed with p53-deficient mice. Whereas the incidence of beta tumor cell apoptosis is unaffected, their proliferation rate is reduced in p53-deficient beta cell tumors in vivo and in cell lines established from these tumors in vitro. Biochemical analyses reveal higher levels of T antigen in wild-type tumor cells as compared to p53-deficient tumor cells. The data indicate that p53 stabilizes SV40 large T antigen, thereby augmenting its oncogenic potential as manifested by increased proliferation rates in wild-type beta tumor cells as compared to p53-deficient beta tumor cells.     

Wild-Type Hras Suppresses the Earliest Stages of Tumorigenesis in a Genetically Engineered Mouse Model of Pancreatic Cancer

Oncogenic, activating mutations in KRAS initiate pancreatic cancer. There are, however, two other Ras family members, Nras and Hras, which can be activated in the presence of oncogenic Kras. The role of these wild-type Ras proteins in cancer remains unclear, as their disruption has been shown to enhance or inhibit tumorigenesis depending upon the context. As pancreatic cancer is critically dependent upon Ras signaling, we tested and now report that loss of Hras increases tumor load and reduces survival in an oncogenic Kras-driven pancreatic adenocarcinoma mouse model. These effects were traced to the earliest stages of pancreatic cancer, suggesting that wild-type Hras may suppress tumor initiation. In normal cells, activated Ras can suppress proliferation through p53-dependent mechanisms. We find that the tumor suppressive effects of Hras are nullified in a homozygous mutant p53 background. As such, loss of wild-type Hras fosters the earliest stages of pancreatic cancer in a p53-dependent manner.     

Inactivation of chk2 and mus81 leads to impaired lymphocytes development, reduced genomic instability, and suppression of cancer

Chk2 is an effector kinase important for the activation of cell cycle checkpoints, p53, and apoptosis in response to DNA damage. Mus81 is required for the restart of stalled replication forks and for genomic integrity. Mus81(Δex3-4/Δex3-4) mice have increased cancer susceptibility that is exacerbated by p53 inactivation. In this study, we demonstrate that Chk2 inactivation impairs the development of Mus81(Δex3-4/Δex3-4) lymphoid cells in a cell-autonomous manner. Importantly, in contrast to its predicted tumor suppressor function, loss of Chk2 promotes mitotic catastrophe and cell death, and it results in suppressed oncogenic transformation and tumor development in Mus81(Δex3-4/Δex3-4) background. Thus, our data indicate that an important role for Chk2 is maintaining lymphocyte development and that dual inactivation of Chk2 and Mus81 remarkably inhibits cancer.