Jesús Gil

Tumour biology: Senescence in premalignant tumours

Oncogene-induced senescence is a cellular response that may be crucial for protection against cancer development, but its investigation has so far been restricted to cultured cells that have been manipulated to overexpress an oncogene. Here we analyse tumours initiated by an endogenous oncogene, ras, and show that senescent cells exist in premalignant tumours but not in malignant ones. Senescence is therefore a defining feature of premalignant tumours that could prove valuable in the diagnosis and prognosis of cancer.

Molecular Interplay of the Noncoding RNA ANRIL and Methylated Histone H3 Lysine 27 by Polycomb CBX7 in Transcriptional Silencing of INK4a

Expression of the INK4b/ARF/INK4a tumor suppressor locus in normal and cancerous cell growth is controlled by methylation of histone H3 at lysine 27 (H3K27me) as directed by the Polycomb group proteins. The antisense noncoding RNA ANRIL of the INK4b/ARF/INK4a locus is also important for expression of the protein-coding genes in cis, but its mechanism has remained elusive. Here we report that chromobox 7 (CBX7) within the polycomb repressive complex 1 binds to ANRIL, and both CBX7 and ANRIL are found at elevated levels in prostate cancer tissues. In concert with H3K27me recognition, binding to RNA contributes to CBX7 function, and disruption of either interaction impacts the ability of CBX7 to repress the INK4b/ARF/INK4a locus and control senescence. Structure-guided analysis reveals the molecular interplay between noncoding RNA and H3K27me as mediated by the conserved chromodomain. Our study suggests a mechanism by which noncoding RNA participates directly in epigenetic transcriptional repression.

Chemokine Signaling via the CXCR2 Receptor Reinforces Senescence

Cells enter senescence, a state of stable proliferative arrest, in response to a variety of cellular stresses, including telomere erosion, DNA damage, and oncogenic signaling, which acts as a barrier against malignant transformation in vivo. To identify genes controlling senescence, we conducted an unbiased screen for small hairpin RNAs that extend the life span of primary human fibroblasts. Here, we report that knocking down the chemokine receptor CXCR2 (IL8RB) alleviates both replicative and oncogene-induced senescence (OIS) and diminishes the DNA-damage response. Conversely, ectopic expression of CXCR2 results in premature senescence via a p53-dependent mechanism. Cells undergoing OIS secrete multiple CXCR2-binding chemokines in a program that is regulated by the NF-kappaB and C/EBPbeta transcription factors and coordinately induce CXCR2 expression. CXCR2 upregulation is also observed in preneoplastic lesions in vivo. These results suggest that senescent cells activate a self-amplifying secretory network in which CXCR2-binding chemokines reinforce growth arrest.

Regulation of the INK4b-ARF-INK4a tumour suppressor locus: all for one or one for all.

The INK4b–ARF–INK4a locus encodes two members of the INK4 family of cyclin-dependent kinase inhibitors, p15INK4b and p16INK4a, and a completely unrelated protein, known as ARF. All three products participate in major tumour suppressor networks that are disabled in human cancer and influence key physiological processes such as replicative senescence, apoptosis and stem-cell self-renewal. Transcription from the locus is therefore kept under strict control. Mounting evidence suggests that although the individual genes can respond independently to positive and negative signals in different contexts, the entire locus might be coordinately suppressed by a cis-acting regulatory domain or by the action of Polycomb group repressor complexes.

Impact of protein kinase PKR in cell biology: from antiviral to antiproliferative action.

SUMMARY The double-stranded RNA-dependent protein kinase PKR is a critical mediator of the antiproliferative and antiviral effects exerted by interferons. Not only is PKR an effector molecule on the cellular response to double-stranded RNA, but it also integrates signals in response to Toll-like receptor activation, growth factors, and diverse cellular stresses. In this review, we provide a detailed picture on how signaling downstream of PKR unfolds and what are the ultimate consequences for the cell fate. PKR activation affects both transcription and translation. PKR phosphorylation of the alpha subunit of eukaryotic initiation factor 2 results in a blockade on translation initiation. However, PKR cannot avoid the translation of some cellular and viral mRNAs bearing special features in their 5′ untranslated regions. In addition, PKR affects diverse transcriptional factors such as interferon regulatory factor 1, STATs, p53, activating transcription factor 3, and NF-κB. In particular, how PKR triggers a cascade of events involving IKK phosphorylation of IκB and NF-κB nuclear translocation has been intensively studied. At the cellular and organism levels PKR exerts antiproliferative effects, and it is a key antiviral agent. A point of convergence in both effects is that PKR activation results in apoptosis induction. The extent and strength of the antiviral action of PKR are clearly understood by the findings that unrelated viral proteins of animal viruses have evolved to inhibit PKR action by using diverse strategies. The case for the pathological consequences of the antiproliferative action of PKR is less understood, but therapeutic strategies aimed at targeting PKR are beginning to offer promising results.

Senescence surveillance of pre-malignant hepatocytes limits liver cancer development.

Upon the aberrant activation of oncogenes, normal cells can enter the cellular senescence program, a state of stable cell-cycle arrest, which represents an important barrier against tumour development in vivo. Senescent cells communicate with their environment by secreting various cytokines and growth factors, and it was reported that this ‘secretory phenotype’ can have pro- as well as anti-tumorigenic effects. Here we show that oncogene-induced senescence occurs in otherwise normal murine hepatocytes in vivo. Pre-malignant senescent hepatocytes secrete chemo- and cytokines and are subject to immune-mediated clearance (designated as ‘senescence surveillance’), which depends on an intact CD4+ T-cell-mediated adaptive immune response. Impaired immune surveillance of pre-malignant senescent hepatocytes results in the development of murine hepatocellular carcinomas (HCCs), thus showing that senescence surveillance is important for tumour suppression in vivo. In accordance with these observations, ras-specific Th1 lymphocytes could be detected in mice, in which oncogene-induced senescence had been triggered by hepatic expression of NrasG12V. We also found that CD4+ T cells require monocytes/macrophages to execute the clearance of senescent hepatocytes. Our study indicates that senescence surveillance represents an important extrinsic component of the senescence anti-tumour barrier, and illustrates how the cellular senescence program is involved in tumour immune surveillance by mounting specific immune responses against antigens expressed in pre-malignant senescent cells.

Senescence impairs successful reprogramming to pluripotent stem cells

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by overexpressing combinations of factors such as Oct4, Sox2, Klf4, and c-Myc. Reprogramming is slow and stochastic, suggesting the existence of barriers limiting its efficiency. Here we identify senescence as one such barrier. Expression of the four reprogramming factors triggers senescence by up-regulating p53, p16(INK4a), and p21(CIP1). Induction of DNA damage response and chromatin remodeling of the INK4a/ARF locus are two of the mechanisms behind senescence induction. Crucially, ablation of different senescence effectors improves the efficiency of reprogramming, suggesting novel strategies for maximizing the generation of iPS cells.

A complex secretory program orchestrated by the inflammasome controls paracrine senescence

Oncogene-induced senescence (OIS) is crucial for tumour suppression. Senescent cells implement a complex pro-inflammatory response termed the senescence-associated secretory phenotype (SASP). The SASP reinforces senescence, activates immune surveillance and paradoxically also has pro-tumorigenic properties. Here, we present evidence that the SASP can also induce paracrine senescence in normal cells both in culture and in human and mouse models of OIS in vivo. Coupling quantitative proteomics with small-molecule screens, we identified multiple SASP components mediating paracrine senescence, including TGF-β family ligands, VEGF, CCL2 and CCL20. Amongst them, TGF-β ligands play a major role by regulating p15INK4b and p21CIP1. Expression of the SASP is controlled by inflammasome-mediated IL-1 signalling. The inflammasome and IL-1 signalling are activated in senescent cells and IL-1α expression can reproduce SASP activation, resulting in senescence. Our results demonstrate that the SASP can cause paracrine senescence and impact on tumour suppression and senescence in vivo.

Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): Mechanism of action

Interferons are a family of cytokines that exerts antiviral, antitumor and immunomodulatory actions by inducing a complex set of proteins. One of the best known IFN-induced protein is the dsRNA-dependent protein kinase (PKR), that mediates both antiviral and anticellular activities. PKR inhibits translation initiation through the phosphorylation of the alpha subunit of the initiation factor eIF-2 (eIF-2α) and also controls the activation of several transcription factors such as NF-κB, p53, or STATs. In addition, PKR mediates apoptosis induced by many different stimuli, such as treatment with LPS, TNF-α, viral infection, or serum starvation. The mechanism of apoptosis induction by PKR involves phosphorylation of eIF-2α and activation of NF-κB. In this way, expression of different genes is regulated by PKR. Among the genes upregulated in response to PKR are Fas, Bax and p53. The pathway of PKR-induced apoptosis involves FADD activation of caspase 8 by a mechanism independent of Fas and TNFR. Since IFNs are used as drugs for different disorders such as viral infection and cancer, understanding the pathway of apoptosis induction triggered by PKR should be useful in the rational design of IFN therapies.

Mouse polycomb proteins bind differentially to methylated histone H3 and RNA and are enriched in facultative heterochromatin.

ABSTRACT The chromodomain (CD) of the Drosophila Polycomb protein exhibits preferential binding affinity for histone H3 when trimethylated at lysine 27. Here we have investigated the five mouse Polycomb homologs known as Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8. Despite a high degree of conservation, the Cbx chromodomains display significant differences in binding preferences. Not all CDs bind preferentially to K27me3; rather, some display affinity towards both histone H3 trimethylated at K9 and H3K27me3, and one CD prefers K9me3. Cbx7, in particular, displays strong affinity for both H3K9me3 and H3K27me3 and is developmentally regulated in its association with chromatin. Cbx7 associates with facultative heterochromatin and, more specifically, is enriched on the inactive X chromosome. Finally, we find that, in vitro, the chromodomain of Cbx7 can bind RNA and that, in vivo, the interaction of Cbx7 with chromatin, and the inactive X chromosome in particular, depends partly on its association with RNA. We propose that the capacity of this mouse Polycomb homolog to associate with the inactive X chromosome, or any other region of chromatin, depends not only on its chromodomain but also on the combination of histone modifications and RNA molecules present at its target sites.