CBX7 and miR‐9 are part of an autoregulatory loop controlling p16INK4a

Polycomb repressive complexes (PRC1 and PRC2) are epigenetic regulators that act in coordination to influence multiple cellular processes including pluripotency, differentiation, cancer and senescence. The role of PRCs in senescence can be mostly explained by their ability to repress the INK4/ARF locus. CBX7 is one of five mammalian orthologues of Drosophila Polycomb that forms part of PRC1. Despite the relevance of CBX7 for regulating senescence and pluripotency, we have a limited understanding of how the expression of CBX7 is regulated. Here we report that the miR‐9 family of microRNAs (miRNAS) downregulates the expression of CBX7. In turn, CBX7 represses miR‐9‐1 and miR‐9‐2 as part of a regulatory negative feedback loop. The miR‐9/CBX7 feedback loop is a regulatory module contributing to induction of the cyclin‐dependent kinase inhibitor (CDKI) p16^INK4a during senescence. The ability of the miR‐9 family to regulate senescence could have implications for understanding the role of miR‐9 in cancer and aging.     

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.     

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.     

The yin and yang of the Cdkn2a locus in senescence and aging

Senescence of cultured cells involves activation of the p19Arf-p53 and the p16Ink4a-Rb tumor suppressor pathways. This, together with the observation that p19Arf and p16Ink4a expression increases with age in many tissues of humans and rodents, led to the speculation that these pathways drive in vivo senescence and natural aging. However, it has been difficult to test this hypothesis using a mammalian model system because inactivation of either of these pathways results in early death from tumors. One approach to bypass this problem would be to inactivate these pathways in a murine segmental progeria model such as mice that express low amounts of the mitotic checkpoint protein BubR1 (BubR1 hypomorphic mice). These mice have a five-fold reduced lifespan and develop a variety of early-aging associated phenotypes including cachetic dwarfism, skeletal muscle degeneration, cataracts, arterial stiffening, (subcutaneous) fat loss, reduced stress tolerance and impaired wound healing. Importantly, BubR1 hypomorphism elevates both p16Ink4a and p19Arf expression in skeletal muscle and fat. Inactivation of p16Ink4a in BubR1 mutant mice delays both cellular senescence and aging specifically in these tissues. Surprisingly, however, inactivation of p19Arf has the opposite effect; it exacerbates in vivo senescence and aging in skeletal muscle and fat. These mouse studies suggest that p16Ink4a is indeed an effector of aging and in vivo senescence, but p19Arf an attenuator. Thus, the role of the p19Arf-p53 pathway in aging and in vivo senescence seems far more complex than previously anticipated.     

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.     

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.     

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.     

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.     

BTG2 antagonizes Pin1 in response to mitogens and telomere disruption during replicative senescence

Cellular senescence limits the replicative capacity of normal cells and acts as an intrinsic barrier that protects against the development of cancer. Telomere shortening–induced replicative senescence is dependent on the ATM‐p53‐p21 pathway but additional genes likely contribute to senescence. Here, we show that the p53‐responsive gene BTG2 plays an essential role in replicative senescence. Similar to p53 and p21 depletion, BTG2 depletion in human fibroblasts leads to an extension of cellular lifespan, and ectopic BTG2 induces senescence independently of p53. The anti‐proliferative function of BTG2 during senescence involves its stabilization in response to telomere dysfunction followed by serum‐dependent binding and relocalization of the cell cycle regulator prolyl isomerase Pin1. Pin1 inhibition leads to senescence in late‐passage cells, and ectopic Pin1 expression rescues cells from BTG2‐induced senescence. The neutralization of Pin1 by BTG2 provides a critical mechanism to maintain senescent arrest in the presence of mitogenic signals in normal primary fibroblasts.     

Extension of replicative lifespan in WI-38 human fibroblasts by dexamethasone treatment is accompanied by suppression of p21 Waf1/Cip1/Sdi1 levels

Numerous studies have shown that supplementation of the growth medium of human fibroblasts with dexamethasone at physiologic concentrations extends replicative lifespan up to 30%. While this extension of lifespan has been used to probe various aspects of the senescent phenotype, no mechanism for the increased lifespan of human fibroblasts grown in the presence of dexamethasone has ever been identified. In the present study we present evidence that the extended lifespan of human lung fibroblasts (WI-38 cells) that occurs when these cells are maintained in culture medium supplemented with dexamethasone is accompanied by a suppression of p21(Waf1/Cip1/Sdi1) levels, which normally increase as these cells enter senescence, while p16(INK4a) levels are unaffected. These results suggest that the delay of senescence in cultures grown in the presence of dexamethasone is due to a suppression of the senescence related increase in p21(Waf1/Cip1/Sdi1). These results are consistent with models of replicative senescence in which p53 and p21(Waf1/Cip1/Sdi1) play a role in the establishment of the senescent arrest.     

Cellular senescence mediated by p16INK4A-coupled miRNA pathways

p16 is a key regulator of cellular senescence, yet the drivers of this stable state of proliferative arrest are not well understood. Here, we identify 22 senescence-associated microRNAs (SA-miRNAs) in normal human mammary epithelial cells. We show that SA-miRNAs-26b, 181a, 210 and 424 function in concert to directly repress expression of Polycomb group (PcG) proteins CBX7, embryonic ectoderm development (EED), enhancer of zeste homologue 2 (EZH2) and suppressor of zeste 12 homologue (Suz12), thereby activating p16. We demonstrate the existence of a tight positive feedback loop in which SA-miRNAs activate and re-enforce the expression of other SA-miRNA members. In contrast, PcG members restrain senescence by epigenetically repressing the expression of these SA-miRNAs. Importantly, loss of p16 leads to repression of SA-miRNA expression, intimately coupling this effector of senescence to the SA-miRNA/PcG self-regulatory loop. Taken together, our findings illuminate an important regulatory axis that underpins the transition from proliferation to cellular senescence.     

Loss of heterozygosity at the Ink4a/Arf locus facilitates Abelson virus transformation of pre-B cells

In many tumor systems, analysis of cells for loss of heterozygosity (LOH) has helped to clarify the role of tumor suppressor genes in oncogenesis. Two important tumor suppressor genes, p53 and the Ink4a/Arf locus, play central roles in the multistep process of Abelson murine leukemia virus (Ab-MLV) transformation. p53 and the p53 regulatory protein, p19Arf, are required for the apoptotic crisis that characterizes the progression of primary transformed pre-B cells to fully malignant cell lines. To search for other tumor suppressor genes which may be involved in the Ab-MLV transformation process, we used endogenous proviral markers and simple-sequence length polymorphism analysis to screen Abelson virus-transformed pre-B cells for evidence of LOH. Our survey reinforces the role of the p53-p19 regulatory pathway in transformation; 6 of 58 cell lines tested had lost sequences on mouse chromosome 4, including the Ink4a/Arf locus. Consistent with this pattern, a high frequency of primary pre-B-cell transformants derived from Ink4a/Arf +/- mice became established cell lines. In addition, half of them retained the single copy of the locus when the transformation process was complete. These data demonstrate that a single copy of the Ink4a/Arf locus is not sufficient to fully mediate the effects of these genes on transformation.