Inhibition of S6 kinase 1 (S6K1) extends lifespan and improves healthspan in mice, but the underlying mechanisms are unclear. Cellular senescence is a stable growth arrest accompanied by an inflammatory senescence-associated secretory phenotype (SASP). Cellular senescence and SASP-mediated chronic inflammation contribute to age-related pathology, but the specific role of S6K1 has not been determined. Here we show that S6K1 deletion does not reduce senescence but ameliorates inflammation in aged mouse livers. Using human and mouse models of senescence, we demonstrate that reduced inflammation is a liver-intrinsic effect associated with S6K deletion. Specifically, we show that S6K1 deletion results in reduced IRF3 activation; impaired production of cytokines, such as IL1 beta; and reduced immune infiltration. Using either liver-specific or myeloid-specific S6K knockout mice, we also demonstrate that reduced immune infiltration and clearance of senescent cells is a hepatocyte-intrinsic phenomenon. Overall, deletion of S6K reduces inflammation in the liver, suggesting that suppression of the inflammatory SASP by loss of S6K could underlie the beneficial effects of inhibiting this pathway on healthspan and lifespan. Inhibition of S6 kinase 1 (S6K1) extends lifespan in mice, but the underlying mechanisms are not fully understood. Here Gallage et al. show that reduction of S6K signaling diminishes inflammation in the aged mouse liver via suppression of the senescence-associated secretory phenotype.

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Senescence is a key barrier to neoplastic transformation. To identify senescence regulators relevant to cancer, we screened a genome-wide shRNA library. Here, we describe exportin 7 (XPO7) as a novel regulator of senescence and validate its function in telomere-induced, replicative, and oncogene-induced senescence (OIS). XPO7 is a bidirectional transporter that regulates the nuclear-cytoplasmic shuttling of a broad range of substrates. Depletion of XPO7 results in reduced levels of TCF3 and an impaired induction of the cyclin-dependent kinase inhibitor p21 CIP1 during OIS. Deletion of XPO7 correlates with poorer overall survival in several cancer types. Moreover, depletion of XPO7 alleviated OIS and increased tumor formation in a mouse model of liver cancer. Our results suggest that XPO7 is a novel tumor suppressor that regulates p21 CIP1 expression to control senescence and tumorigenesis.

The European H5N8 highly pathogenic avian influenza virus (HPAIV) epizootic during 2016-2017 resulted in both wild bird and poultry deaths throughout the EU. This in vivo study investigated the potential for indirect infection of naïve birds through contaminated drinking water or feed, to assess potential disease incursion into a high biosecurity commercial floor housed setting. Three-week-old Ross 308 chickens were exposed to H5N8 A/wigeon/Wales/52833/2016 (H5N8-2016) virus at a high (1 x 106 EID50/ml) or low (1 x 104 EID50/ml) dose, in either drinking water or feed for a 24 hour period. Chickens directly-infected with a high dose of H5N8-2016 (intra-nasal) acted as positive controls. Viral shedding, environmental contamination and clinical signs were monitored for ten days post infection (dpi). All directly-infected birds shed virus and were humanely terminated at 3 dpi. Immunohistochemical analysis of nasal epithelium and caecal tonsil lymphoid tissue, obtained at post mortem from directly-infected chickens (2 dpi), showed the presence of influenza antigen in both tissues. Only birds exposed to high dose virus in drinking water, shed virus and showed clinical disease presentation (67% mortality). Interestingly low levels of antigen were detected in the nasal epithelium, whereas higher levels were detected in the caecal tonsil. All surviving chickens from each group, remained uninfected and did not seroconvert. Our findings suggest virus bio availability in different substrates is variable (feed and water) and possible routes of viral contamination leading to disease ingress at poultry premises may have different outcomes including disease presentation.

Previously we successful infected turkeys with the China-origin H7N9 low pathogenicity avian influenza virus (LPAIV, A/Anhui/1/13, referred to as ‘wild-type’ (Wt)) which successfully transmitted to contact turkeys with virulent outcomes, highly unusual for LPAIV infection, particularly as the LPAIV cleavage site remained unchanged in all experiments. Sequencing of progeny viruses revealed consistent emergence of the L226Q polymorphism in the HA gene, termed the ‘turkey-adapted’ (ty-ad) virus. Ty-ad and Wt were used to compare the epizootic risk posed by both H7N9 LPAIVs in turkeys and to explore the mechanisms which underpins any differences. The Wt and ty-ad viruses robustly infected inoculated and contact turkeys, producing similar shedding titres. However, the ty-ad virus was more pathogenic than the Wt virus in directly-infected and contact turkeys, causing 100 % (Wt) compared to 16 % (ty-ad) survival. The ty-ad virus was detected in broader range of turkey organs, and at higher titre, compared to the Wt variant. This contrasted with pathogenicity and tissue-tropism observations for both viruses in chickens. The wt and ty-ad viruses did not replicate without trypsin in vitro, affirming a typical LPAIV phenotype. The L226Q polymorphism is known to alter receptor binding, with key differences in receptor distribution between turkey and chicken tissues observed. Replication kinetics differences in a range of avian cells will be reported for both viruses. Consequently, if this ty-ad variant were to arise more frequently in nature, it would pose an increased virulent risk to turkeys. It is therefore important to maintain surveillance and understanding of China-origin H7N9 viruses.

Swine influenza A virus (swIAV) causes respiratory disease and productivity loss in pigs. Swine ‘flu viruses have been known to be both zoonotic and reverse zoonotic and they contain genes of swine, avian(av) and human(hu) origin. Surveillance of swIAV subtypes is important as genotypes/phenotypes are fluid and impact with respect to epidemiology, vaccination, pig welfare, veterinary and public health. Three sub-types (H1avN1, H1N1pdm09, H1huN2) are currently found in pigs from Great Britain (GB), plus H3huN2 in Europe and their reassortants. Screening of candidate samples is carried out by RRT-PCR assays – generic detection of swIAV (M gene) followed by a specific RRT-PCR for H1N1pdm09 (HA gene), a suite of RRT-PCR assays for sub-typing (HA and NA genes) and a (differential) RRT-PCR to specifically identify reassortant swIAVs that incorporate the pandemic 2009 internal gene cassette (NP gene). Subtyping assays, conventional and/or molecular, are carried out on virus isolation-positive and –negative (RNA only) samples from clinical material (respiratory tissue and/or nasal swabs). Since 2009, the number of swIAV has expanded with the H1N1pdm09 isolates reassorting with the traditional subtypes. Many European variants arose (>25) of which some have become established – in GB including H1huN2/pdm (since 2010), and H1avN1/pdm (since 2012), and in Belgium the traditional isolates were detected plus H1pdmN1/pdm and H3huN2/pdm reassortants. PCR subtyping (2012 onwards ∼130 from GB and ∼40 from BE/NL), wholegenome sequencing and bioinformatics analysis of these isolates facilitate further diagnostic improvements and assessment of zoonotic pandemic potential (in silico and in vivo).

With the wild bird migratory season reaching its peak, tens of thousands of wild waterfowl have been reaching UK shores from the Baltic States and further afield. It has been established that migratory birds play a role in the spread of highly pathogenic avian influenza (HPAI).1 The detection of H5N8 HPAI cases in wild birds, and outbreaks in poultry and captive birds across Europe during the last migratory season broadly followed this migration trajectory.2Cases of H5N8 HPAI in 2016/17 across Europe peaked in the early months of 2017. In the UK, a total of 13 cases were identified in poultry and gamebirds (six cases in back yard flocks, seven in commercial premises). In addition, 45 H5N8 positive wild birds (mostly waterfowl) were identified, which were submitted to the APHA via the wild bird surveillance scheme. Epidemiological analysis concluded that, apart from one cluster of related premises, each case was a …

With the wild bird migratory season reaching its peak, tens of thousands of wild waterfowl have been reaching UK shores from the Baltic States and further afield. It has been established that migratory birds play a role in the spread of highly pathogenic avian influenza (HPAI).1 The detection of H5N8 HPAI cases in wild birds, and outbreaks in poultry and captive birds across Europe during the last migratory season broadly followed this migration trajectory.2 Cases of H5N8 HPAI in 2016/17 across Europe peaked in the early months of 2017. In the UK, a total of 13 cases were identified in poultry and gamebirds (six cases in back yard flocks, seven in commercial premises). In addition, 45 H5N8 positive wild birds (mostly waterfowl) were identified, which were submitted to the APHA via the wild bird surveillance scheme. Epidemiological analysis concluded that, apart from one cluster of related premises, each case was a …

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.

Cellular senescence, the stable cell cycle arrest elicited by various forms of stress, is an important facet of tumor suppression. Although much is known about the key players in the implementation of senescence, including the pRb and p53 axes and the cyclin dependent kinase inhibitors p16(INK4a) and p21(CIP1), many details remain unresolved. In studying conditional senescence in human fibroblasts that express a temperature sensitive SV40 large T-antigen (T-Ag), we uncovered an unexpected role for CDK4. At the permissive temperature, where pRb and p53 are functionally compromised by T-Ag, cyclin D-CDK4 complexes are disrupted by the high p16(INK4a) levels and reduced expression of p21(CIP1). In cells arrested at the non-permissive temperature, p21(CIP1) promotes reassembly of cyclin D-CDK4 yet pRb is in a hypo-phosphorylated state, consistent with cell cycle arrest. In exploring whether the reassembled cyclin D-CDK4-p21 complexes are functional, we found that shRNA-mediated knockdown or chemical inhibition of CDK4 prevented the increase in cell size associated with the senescent phenotype by allowing the cells to arrest in G1 rather than G2/M. The data point to a role for CDK4 kinase activity in a G2 checkpoint that contributes to senescence.

BACKGROUND:Polycomb group proteins form multicomponent complexes that are important for establishing lineage-specific patterns of gene expression. Mammalian cells encode multiple permutations of the prototypic Polycomb repressive complex 1 (PRC1) with little evidence for functional specialization. An aim of this study is to determine whether the multiple orthologs that are co-expressed in human fibroblasts act on different target genes and whether their genomic location changes during cellular senescence.RESULTS:Deep sequencing of chromatin immunoprecipitated with antibodies against CBX6, CBX7, CBX8, RING1 and RING2 reveals that the orthologs co-localize at multiple sites. PCR-based validation at representative loci suggests that a further six PRC1 proteins have similar binding patterns. Importantly, sequential chromatin immunoprecipitation with antibodies against different orthologs implies that multiple variants of PRC1 associate with the same DNA. At many loci, the binding profiles have a distinctive architecture that is preserved in two different types of fibroblast. Conversely, there are several hundred loci at which PRC1 binding is cell type-specific and, contrary to expectations, the presence of PRC1 does not necessarily equate with transcriptional silencing. Interestingly, the PRC1 binding profiles are preserved in senescent cells despite changes in gene expression.CONCLUSIONS:The multiple permutations of PRC1 in human fibroblasts congregate at common rather than specific sites in the genome and with overlapping but distinctive binding profiles in different fibroblasts. The data imply that the effects of PRC1 complexes on gene expression are more subtle than simply repressing the loci at which they bind.

A growing body of evidence suggests that Polycomb group (PcG) proteins, key regulators of lineage specific gene expression, also participate in the repair of DNA double-strand breaks (DSBs) but evidence for direct recruitment of PcG proteins at specific breaks remains limited. Here we explore the association of Polycomb repressive complex 1 (PRC1) components with DSBs generated by inducible expression of the AsiSI restriction enzyme in normal human fibroblasts. Based on immunofluorescent staining, the co-localization of PRC1 proteins with components of the DNA damage response (DDR) in these primary cells is unconvincing. Moreover, using chromatin immunoprecipitation and deep sequencing (ChIP-seq), which detects PRC1 proteins at common sites throughout the genome, we did not find evidence for recruitment of PRC1 components to AsiSI-induced DSBs. In contrast, the S2056 phosphorylated form of DNA-PKcs and other DDR proteins were detected at a subset of AsiSI sites that are predominantly at the 5' ends of transcriptionally active genes. Our data question the idea that PcG protein recruitment provides a link between DSB repairs and transcriptional repression.

The nuclear receptor NR2E1 (also known as TLX or tailless) controls the self-renewal of neural stem cells (NSCs) and has been implied as an oncogene which initiates brain tumors including glioblastomas. Despite NR2E1 regulating targets like p21(CIP1) or PTEN we still lack a full explanation for its role in NSC self-renewal and tumorigenesis. We know that polycomb repressive complexes also control stem cell self-renewal and tumorigenesis, but so far, no formal connection has been established between NR2E1 and PRCs. In a screen for transcription factors regulating the expression of the polycomb protein CBX7, we identified NR2E1 as one of its more prominent regulators. NR2E1 binds at the CBX7 promoter, inducing its expression. Notably CBX7 represses NR2E1 as part of a regulatory loop. Ectopic NR2E1 expression inhibits cellular senescence, extending cellular lifespan in fibroblasts via CBX7-mediated regulation of p16(INK4a) and direct repression of p21(CIP1). In addition NR2E1 expression also counteracts oncogene-induced senescence. The importance of NR2E1 to restrain senescence is highlighted through the process of knocking down its expression, which causes premature senescence in human fibroblasts and epithelial cells. We also confirmed that NR2E1 regulates CBX7 and restrains senescence in NSCs. Finally, we observed that the expression of NR2E1 directly correlates with that of CBX7 in human glioblastoma multiforme. Overall we identified control of senescence and regulation of polycomb action as two possible mechanisms that can join those so far invoked to explain the role of NR2E1 in control of NSC self-renewal and cancer.

Abstract The cyclin-dependent kinase (CDK) inhibitors, p18INK4c and p16INK4a, both have the credentials of tumor suppressors in human cancers and mouse models. For p16INK4a, the underlying rationale is its role in senescence, but the selective force for inactivation of p18INK4c in incipient cancer cells is less clear. Here, we show that in human fibroblasts undergoing replicative or oncogene-induced senescence, there is a marked decline in the levels of p18INK4c protein and RNA, which mirrors the accumulation of p16INK4a. Downregulation of INK4c is not dependent on p16INK4a, and RAS can promote the loss of INK4c without cell-cycle arrest. Downregulation of p18INK4c correlates with reduced expression of menin and E2F1 but is unaffected by acute cell-cycle arrest or inactivation of the retinoblastoma protein (pRb). Collectively, our data question the idea that p18INK4c acts as a backup for loss of p16INK4a and suggest that the apparent activation of p18INK4c in some settings represents delayed senescence rather than increased expression. We propose that the contrasting behavior of the two very similar INK4 proteins could reflect their respective roles in senescence versus differentiation. Cancer Res; 72(1); 165–75. ©2011 AACR.

The cyclin-dependent kinase (CDK) inhibitors, p18 and p16, both have the credentials of tumor suppressors in human cancers andmousemodels. For p16, the underlying rationale is its role in senescence, but the selective force for inactivation of p18 in incipient cancer cells is less clear. Here, we show that in human fibroblasts undergoing replicative or oncogene-induced senescence, there is amarked decline in the levels of p18 protein and RNA, which mirrors the accumulation of p16. Downregulation of INK4c is not dependent on p16, and RAS can promote the loss of INK4c without cell-cycle arrest. Downregulation of p18 correlates with reduced expression of menin and E2F1 but is unaffected by acute cell-cycle arrest or inactivation of the retinoblastoma protein (pRb). Collectively, our data question the idea that p18 acts as a backup for loss of p16 and suggest that the apparent activation of p18 in some settings represents delayed senescence rather than increased expression. We propose that the contrasting behavior of the two very similar INK4 proteins could reflect their respective roles in senescence versus differentiation. Cancer Res; 72(1); 165–75. 2011 AACR.