Opposing roles for p16Ink4a and p19Arf in senescence and ageing caused by BubR1 insufficiency

Expression of p16(Ink4a) and p19(Arf) increases with age in both rodent and human tissues. However, whether these tumour suppressors are effectors of ageing remains unclear, mainly because knockout mice lacking p16(Ink4a) or p19(Arf) die early of tumours. Here, we show that skeletal muscle and fat, two tissues that develop early ageing-associated phenotypes in response to BubR1 insufficiency, have high levels of p16(Ink4a) and p19(Arf). Inactivation of p16(Ink4a) in BubR1-insufficient mice attenuates both cellular senescence and premature ageing in these tissues. Conversely, p19(Arf) inactivation exacerbates senescence and ageing in BubR1 mutant mice. Thus, we identify BubR1 insufficiency as a trigger for activation of the Cdkn2a locus in certain mouse tissues, and demonstrate that p16(Ink4a) is an effector and p19(Arf) an attenuator of senescence and ageing in these tissues.     

Losses of both products of the Cdkn2a/Arf locus contribute to asbestos-induced mesothelioma development and cooperate to accelerate tumorigenesis

The CDKN2A/ARF locus encompasses overlapping tumor suppressor genes p16(INK4A) and p14(ARF), which are frequently co-deleted in human malignant mesothelioma (MM). The importance of p16(INK4A) loss in human cancer is well established, but the relative significance of p14(ARF) loss has been debated. The tumor predisposition of mice singly deficient for either Ink4a or Arf, due to targeting of exons 1α or 1β, respectively, supports the idea that both play significant and nonredundant roles in suppressing spontaneous tumors. To further test this notion, we exposed Ink4a(+/-) and Arf(+/-) mice to asbestos, the major cause of MM. Asbestos-treated Ink4a(+/-) and Arf(+/-) mice showed increased incidence and shorter latency of MM relative to wild-type littermates. MMs from Ink4a(+/-) mice exhibited biallelic inactivation of Ink4a, loss of Arf or p53 expression and frequent loss of p15(Ink4b). In contrast, MMs from Arf(+/-) mice exhibited loss of Arf expression, but did not require loss of Ink4a or Ink4b. Mice doubly deficient for Ink4a and Arf, due to deletion of Cdkn2a/Arf exon 2, showed accelerated asbestos-induced MM formation relative to mice deficient for Ink4a or Arf alone, and MMs exhibited biallelic loss of both tumor suppressor genes. The tumor suppressor function of Arf in MM was p53-independent, since MMs with loss of Arf retained functional p53. Collectively, these in vivo data indicate that both CDKN2A/ARF gene products suppress asbestos carcinogenicity. Furthermore, while inactivation of Arf appears to be crucial for MM pathogenesis, the inactivation of both p16(Ink4a) and p19(Arf) cooperate to accelerate asbestos-induced tumorigenesis.     

Anti-aging activity of the Ink4/Arf locus

The proteins encoded by the Ink4/Arf locus, p16^Ink4a, p19^Arf and p15^Ink4b are major tumour suppressors that oppose aberrant mitogenic signals. The expression levels of the locus are progressively increased during aging and genome-wide association studies have linked the locus to a number of aging-associated diseases and frailty in humans. However, direct measurement of the global impact of the Ink4/Arf locus on organismal aging and longevity was lacking. In this work, we have examined the fertility, cancer susceptibility, aging and longevity of mice genetically modified to carry one ( Ink4/Arf -tg) or two ( Ink4/Arf -tg/tg) intact additional copies of the locus. First, increased gene dosage of Ink4/Arf impairs the production of male germ cells, and in the case of Ink4/Arf -tg/tg mice results in a Sertoli cell-only-like syndrome and a complete absence of sperm. Regarding cancer, there is a lower incidence of aging-associated cancer proportional to the Ink4/Arf gene dosage. Interestingly, increased Ink4/Arf gene dosage resulted in lower scores in aging markers and in extended median longevity. The increased survival was also observed in cancer-free mice indicating that cancer protection and delayed aging are separable activities of the Ink4/Arf locus. In contrast to these results, mice carrying one or two additional copies of the p53 gene ( p53 -tg and p53 -tg/tg) had a normal longevity despite their increased cancer protection. We conclude that the Ink4/Arf locus has a global anti-aging effect, probably by favouring quiescence and preventing unnecessary proliferation.     

p16(Ink4a) interferes with Abelson virus transformation by enhancing apoptosis

Pre-B-cell transformation by Abelson virus (Ab-MLV) is a multistep process in which primary transformants are stimulated to proliferate but subsequently undergo crisis, a period of erratic growth marked by high levels of apoptosis. Inactivation of the p53 tumor suppressor pathway is an important step in this process and can be accomplished by mutation of p53 or down-modulation of p19(Arf), a p53 regulatory protein. Consistent with these data, pre-B cells from either p53 or Ink4a/Arf null mice bypass crisis. However, the Ink4a/Arf locus encodes both p19(Arf) and a second tumor suppressor, p16(Ink4a), that blocks cell cycle progression by inhibiting Cdk4/6. To determine if p16(Ink4a) plays a role in Ab-MLV transformation, primary transformants derived from Arf(-/-) and p16(Ink4a(-/-)) mice were compared. A fraction of those derived from Arf(-/-) animals underwent crisis, and even though all p16(Ink4a(-/-)) primary transformants experienced crisis, these cells became established more readily than cells derived from +/+ mice. Analyses of Ink4a/Arf(-/-) cells infected with a virus that expresses both v-Abl and p16(Ink4a) revealed that p16(Ink4a) expression does not alter cell cycle profiles but does increase the level of apoptosis in primary transformants. These results indicate that both products of the Ink4a/Arf locus influence Ab-MLV transformation and reveal that in addition to its well-recognized effects on the cell cycle, p16(Ink4a) can suppress transformation by inducing apoptosis.     

Obligate Roles for p16Ink4a and p19Arf-p53 in the Suppression of Murine Pancreatic Neoplasia

Epithelial tumors of the pancreas exhibit a wide spectrum of histologies with varying propensities for metastasis and tissue invasion. The histogenic relationship among these tumor types is not well established; moreover, the specific role of genetic lesions in the progression of these malignancies is largely undefined. Transgenic mice with ectopic expression of transforming growth factor alpha (TGF-alpha) in the pancreatic acinar cells develop tubular metaplasia, a potential premalignant lesion of the pancreatic ductal epithelium. To evaluate the cooperative interactions between TGF-alpha and signature mutations in pancreatic tumor genesis and progression, TGFalpha transgenic mice were crossed onto Ink4a/Arf and/or p53 mutant backgrounds. These compound mutant mice developed a novel pancreatic neoplasm, serous cystadenoma (SCA), presenting as large epithelial tumors bearing conspicuous gross and histological resemblances to their human counterpart. TGFalpha animals heterozygous for both the Ink4a/Arf and the p53 mutation showed a dramatically increased incidence of SCA, indicating synergistic interaction of these alleles. Inactivation of p16(Ink4a) by loss of heterozygosity, intragenic mutation, or promoter hypermethylation was a common feature in these SCAs, and correspondingly, none of the tumors expressed wild-type p16(Ink4a). All tumors sustained loss of p53 or Arf, generally in a mutually exclusive fashion. The tumor incidence data and molecular profiles establish a pathogenic role for the dual inactivation of p16(Ink4a) and p19(Arf)-p53 in the development of SCA in mice, demonstrating that p16(Ink4a) is a murine tumor suppressor. This genetically defined model provides insights into the molecular pathogenesis of SCA and serves as a platform for dissection of cell-specific programs of epithelial tumor suppression.     

Loss of miRNA biogenesis induces p19Arf-p53 signaling and senescence in primary cells

Dicer, an enzyme involved in microRNA (miRNA) maturation, is required for proper cell differentiation and embryogenesis in mammals. Recent evidence indicates that Dicer and miRNA may also regulate tumorigenesis. To better characterize the role of miRNA in primary cell growth, we generated Dicer-conditional mice. Ablation of Dicer and loss of mature miRNAs in embryonic fibroblasts up-regulated p19(Arf) and p53 levels, inhibited cell proliferation, and induced a premature senescence phenotype that was also observed in vivo after Dicer ablation in the developing limb and in adult skin. Furthermore, deletion of the Ink4a/Arf or p53 locus could rescue fibroblasts from premature senescence induced by Dicer ablation. Although levels of Ras and Myc oncoproteins appeared unaltered, loss of Dicer resulted in increased DNA damage and p53 activity in these cells. These results reveal that loss of miRNA biogenesis activates a DNA damage checkpoint, up-regulates p19(Arf)-p53 signaling, and induces senescence in primary cells.     

Nucleolar Arf sequesters Mdm2 and activates p53

The Ink4/Arf locus encodes two tumour-suppressor proteins, p16Ink4a and p19Arf, that govern the antiproliferative functions of the retinoblastoma and p53 proteins, respectively. Here we show that Arf binds to the product of the Mdm2 gene and sequesters it into the nucleolus, thereby preventing negative-feedback regulation of p53 by Mdm2 and leading to the activation of p53 in the nucleoplasm. Arf and Mdm2 co-localize in the nucleolus in response to activation of the oncoprotein Myc and as mouse fibroblasts undergo replicative senescence. These topological interactions of Arf and Mdm2 point towards a new mechanism for p53 activation.     

Early aging-associated phenotypes in Bub3/Rae1 haploinsufficient mice

Aging is a highly complex biological process that is believed to involve multiple mechanisms. Mice that have small amounts of the mitotic checkpoint protein BubR1 age much faster than normal mice, but whether other mitotic checkpoint genes function to prevent the early onset of aging is unknown. In this study, we show that several aging-associated phenotypes appear early in mice that are double haploinsufficient for the mitotic checkpoint genes Bub3 and Rae1 but not in mice that are single haploinsufficient for these genes. Mouse embryonic fibroblasts (MEFs) from Bub3/Rae1 haploinsufficient mice undergo premature senescence and accumulate high levels of p19, p53, p21, and p16, whereas MEFs from single haploinsufficient mice do not. Furthermore, although BubR1 hypomorphic mice have less aneuploidy than Bub3/Rae1 haploinsufficient mice, they age much faster. Our findings suggest that early onset of aging-associated phenotypes in mice with mitotic checkpoint gene defects is linked to cellular senescence and activation of the p53 and p16 pathways rather than to aneuploidy.     

Polycomb CBX7 has a unifying role in cellular lifespan

In contrast to cancer cells and embryonic stem cells, the lifespan of primary human cells is finite. After a defined number of population doublings, cells enter in an irreversible growth-arrested state termed replicative senescence. Mutations of genes involved in immortalization can contribute to cancer. In a genetic screen for cDNAs bypassing replicative senescence of normal human prostate epithelial cells (HPrEC), we identified CBX7, a gene that encodes a Polycomb protein, as shown by sequence homology, its interaction with Ring1 and its localization to nuclear Polycomb bodies. CBX7 extends the lifespan of a wide range of normal human cells and immortalizes mouse fibroblasts by downregulating expression of the Ink4a/Arf locus. CBX7 does not inter-function or colocalize with Bmi1, and both can exert their actions independently of each other as shown by reverse genetics. CBX7 expression is downregulated during replicative senescence and its ablation by short-hairpin RNA (shRNA) treatment inhibited growth of normal cells though induction of the Ink4a/Arf locus. Taken together, these data show that CBX7 controls cellular lifespan through regulation of both the p16(Ink4a)/Rb and the Arf/p53 pathways.     

Similar Tumor Suppressor Gene Alteration Profiles in Asbestos-Induced Murine and Human Mesothelioma

The status of tumor suppressor genes (TSGs) relevant to human malignant mesothelioma (HMM) pathogenesis was examined in cultures of mesothelioma cells from tumoral ascites developed in mice exposed to asbestos (asb) fibers. The status of the respective hortologous human genes was also investigated in 12 HMM cell cultures. Eleven primary cultures from mice hemizygous for N?2 (asb-Nf2KO3/+) and 4 wild type counterparts (asb-Nf2+/+) were analyzed for mutations in Nf2, p16/Cdkn2a, p19/Arf and Trp53 genes and protein expression of p15/Cdkn2b and Cdk4. TSG alterations in both mouse and human mesothelioma cells consisted in frequent inactivation of p16/Cdkn2a, p19/Arf (or P14/ARF) and p15/Cdkn2b, co-inactivation of p16/Cdkn2a and p15/Cdkn2b and low rate of Trp53 mutations in both asb-Nf2KO3/+ and asb-Nf2+/+ mesothelioma cells. In both mouse and human mesothelioma cells, inactivation of the hortologous genes p16/Cdkn2a or P16/CDKN2A was due to deletions at the Ink4/Arf locus encompassing p19/Arf or P14/ARF, respectively. Loss of heterozygosity at the Nf2 locus was detected in 10 of 11 asb-Nf2KO3/+ cultures and Nf2 gene rearrangement in one asb-Nf2+/+ culture. These data show that the profile of TSG alterations in asbestos-induced mesothelioma is similar in mice and humans. Thus, the mouse mesothelioma model could be useful for human risk assessment, taking into account interindividual variations in genetic sensitivity to carcinogens.     

Synergism between INK4a/ARF inactivation and aberrant HGF/SF signaling in rhabdomyosarcomagenesis

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children, yet molecular events associated with the genesis and progression of this potentially fatal disease are largely unknown. For the molecules and pathways that have been implicated, genetic validation has been impeded by lack of a mouse model of RMS. Here we show that simultaneous loss of Ink4a/Arf function and disruption of c-Met signaling in Ink4a/Arf(-/-) mice transgenic for hepatocyte growth factor/scatter factor (HGF/SF) induces RMS with extremely high penetrance and short latency. In cultured myoblasts, c-Met activation and Ink4a/Arf loss suppress myogenesis in an additive fashion. Our data indicate that human c-MET and INK4a/ARF, situated at the nexus of pathways regulating myogenic growth and differentiation, represent critical targets in RMS pathogenesis. The marked synergism in mice between aberrant c-Met signaling and Ink4a/Arf inactivation, lesions individually implicated in human RMS, suggests a therapeutic combination to combat this devastating childhood cancer.     

Increased gene dosage of Ink4/Arf and p53 delays age‐associated central nervous system functional decline

The impairment of the activity of the brain is a major feature of aging, which coincides with a decrease in the function of neural stem cells. We have previously shown that an extra copy of regulated Ink4/Arf and p53 activity, in s‐Ink4/Arf/p53 mice, elongates lifespan and delays aging. In this work, we examined the physiology of the s‐Ink4/Arf/p53 brain with aging, focusing on the neural stem cell (NSC) population. We show that cells derived from old s‐Ink4/Arf/p53 mice display enhanced neurosphere formation and self‐renewal activity compared with wt controls. This correlates with augmented expression of Sox2, Sox9, Glast, Ascl1, and Ars2 NSC markers in the subventricular zone (SVZ) and in the subgranular zone of the dentate gyrus (DG) niches. Furthermore, aged s‐Ink4/Arf/p53 mice express higher levels of Doublecortin and PSA‐NCAM (neuroblasts) and NeuN (neurons) in the olfactory bulbs (OB) and DG, indicating increased neurogenesis in vivo. Finally, aged s‐Ink4/Arf/p53 mice present enhanced behavioral and neuromuscular coordination activity. Together, these findings demonstrate that increased but regulated Ink4/Arf and p53 activity ameliorates age‐related deterioration of the central nervous system activity required to maintain the stem cell pool, providing a mechanism not only for the extended lifespan but also for the health span of these mice.     

Senescence promotes in vivo reprogramming through p16INK4a and IL‐6

Cellular senescence is a damage response aimed to orchestrate tissue repair. We have recently reported that cellular senescence, through the paracrine release of interleukin‐6 (IL6) and other soluble factors, strongly favors cellular reprogramming by Oct4, Sox2, Klf4, and c‐Myc (OSKM) in nonsenescent cells. Indeed, activation of OSKM in mouse tissues triggers senescence in some cells and reprogramming in other cells, both processes occurring concomitantly and in close proximity. In this system, Ink4a/Arf‐null tissues cannot undergo senescence, fail to produce IL6, and cannot reprogram efficiently; whereas p53‐null tissues undergo extensive damage and senescence, produce high levels of IL6, and reprogram efficiently. Here, we have further explored the genetic determinants of in vivo reprogramming. We report that Ink4a, but not Arf, is necessary for OSKM‐induced senescence and, thereby, for the paracrine stimulation of reprogramming. However, in the absence of p53, IL6 production and reprogramming become independent of Ink4a, as revealed by the analysis of Ink4a/Arf/p53 deficient mice. In the case of the cell cycle inhibitor p21, its protein levels are highly elevated upon OSKM activation in a p53‐independent manner, and we show that p21‐null tissues present increased levels of senescence, IL6, and reprogramming. We also report that Il6‐mutant tissues are impaired in undergoing reprogramming, thus reinforcing the critical role of IL6 in reprogramming. Finally, young female mice present lower efficiency of in vivo reprogramming compared to male mice, and this gender difference disappears with aging, both observations being consistent with the known anti‐inflammatory effect of estrogens. The current findings regarding the interplay between senescence and reprogramming may conceivably apply to other contexts of tissue damage.     

Murine mesenchymal cells that express elevated levels of the CDK inhibitor p16(Ink4a) in vivo are not necessarily senescent

Age-related health decline has been attributed to the accumulation of senescent cells recognized in vivo by p16(Ink4a) expression. The pharmacological elimination of p16(Ink4a)-positive cells from the tissues of mice was shown to extend a healthy lifespan. Here, we describe a population of mesenchymal cells isolated from mice that are highly p16(INK4a)-positive are proficient in proliferation but lack other properties of cellular senescence. These data, along with earlier reports on p16(Ink4a)-positive macrophages, indicate that p16(Ink4a)-positive and senescent cell populations only partially intersect, therefore, extending the list of potential cellular targets for anti- aging therapies.     

Tissue specificity of senescent cell accumulation during physiologic and accelerated aging of mice

Senescent cells accumulate with age in vertebrates and promote aging largely through their senescence‐associated secretory phenotype (SASP). Many types of stress induce senescence, including genotoxic stress. ERCC1‐XPF is a DNA repair endonuclease required for multiple DNA repair mechanisms that protect the nuclear genome. Humans or mice with reduced expression of this enzyme age rapidly due to increased levels of spontaneous, genotoxic stress. Here, we asked whether this corresponds to an increased level of senescent cells. p16^Ink4a and p21^Cip1 mRNA were increased ~15‐fold in peripheral lymphocytes from 4‐ to 5‐month‐old Ercc1^?/? and 2.5‐year‐old wild‐type (WT) mice, suggesting that these animals exhibit a similar biological age. p16^Ink4a and p21^Cip1 mRNA were elevated in 10 of 13 tissues analyzed from 4‐ to 5‐month‐old Ercc1^?/? mice, indicating where endogenous DNA damage drives senescence in vivo. Aged WT mice had similar increases of p16^Ink4a and p21^Cip1 mRNA in the same 10 tissues as the mutant mice. Senescence‐associated β–galactosidase activity and p21^Cip1 protein also were increased in tissues of the progeroid and aged mice, while Lamin B1 mRNA and protein levels were diminished. In Ercc1^?/Δ mice with a p16^Ink4a luciferase reporter, bioluminescence rose steadily with age, particularly in lung, thymus, and pancreas. These data illustrate where senescence occurs with natural and accelerated aging in mice and the relative extent of senescence among tissues. Interestingly, senescence was greater in male mice until the end of life. The similarities between Ercc1^?/? and aged WT mice support the conclusion that the DNA repair‐deficient mice accurately model the age‐related accumulation of senescent cells, albeit six‐times faster.     

Reversible cell cycle inhibition and premature aging features imposed by conditional expression of p16Ink4a

The cyclin‐dependent kinase (Cdk) inhibitor p16^Ink4a (p16) is a canonical mediator of cellular senescence and accumulates in aging tissues, where it constrains proliferation of some progenitor cells. However, whether p16 induction in tissues is sufficient to inhibit cell proliferation, mediate senescence, and/or impose aging features has remained unclear. To address these issues, we generated transgenic mice that permit conditional p16 expression. Broad induction at weaning inhibited proliferation of intestinal transit‐amplifying and Lgr5+ stem cells and rapidly imposed features of aging, including hair loss, skin wrinkling, reduced body weight and subcutaneous fat, an increased myeloid fraction in peripheral blood, poor dentition, and cataracts. Aging features were observed with multiple combinations of p16 transgenes and transactivators and were largely abrogated by a germline Cdk4 R24C mutation, confirming that they reflect Cdk inhibition. Senescence markers were not found, and de‐induction of p16, even after weeks of sustained expression, allowed rapid recovery of intestinal cell proliferation and reversal of aging features in most mice. These results suggest that p16‐mediated inhibition of Cdk activity is sufficient to inhibit cell proliferation and impose aging features in somatic tissues of mammals and that at least some of these aging features are reversible.