Reciprocal regulation of p21 and Chk1 controls the cyclin D1-RB pathway to mediate senescence onset after G2 arrest.

Senescence is an irreversible withdrawal from cell proliferation that can be initiated after DNA damage-induced cell cycle arrest in G2 phase to prevent genomic instability. Senescence onset in G2 requires p53 (also known as TP53) and retinoblastoma protein (RB, also known as RB1) family tumour suppressors, but how they are regulated to convert a temporary cell cycle arrest into a permanent one remains unknown. Here, we show that a previously unrecognised balance between the cyclin-dependent kinase (CDK) inhibitor p21 and the checkpoint kinase Chk1 controls cyclin D-CDK activity during G2 arrest. In non-transformed cells, p21 activates RB in G2 by inhibiting cyclin D1 complexed with CDK2 or CDK4. The resulting G2 exit, which precedes the appearance of senescence markers, is associated with a mitotic bypass, Chk1 downregulation and reduction in the number of DNA damage foci. In p53/RB-proficient cancer cells, a compromised G2 exit correlates with sustained Chk1 activity, delayed p21 induction, untimely cyclin E1 re-expression and genome reduplication. Conversely, Chk1 depletion promotes senescence by inducing p21 binding to cyclin D1- and cyclin E1-CDK complexes and downregulating CDK6, whereas knockdown of the checkpoint kinase Chk2 enables RB phosphorylation and delays G2 exit. In conclusion, p21 and Chk2 oppose Chk1 to maintain RB activity, thus promoting the onset of senescence induced by DNA damage in G2.

Mechanisms and Regulation of Cellular Senescence.

Cellular senescence entails a state of an essentially irreversible proliferative arrest in which cells remain metabolically active and secrete a range of pro-inflammatory and proteolytic factors as part of the senescence-associated secretory phenotype. There are different types of senescent cells, and senescence can be induced in response to many DNA damage signals. Senescent cells accumulate in different tissues and organs where they have distinct physiological and pathological functions. Despite this diversity, all senescent cells must be able to survive in a nondividing state while protecting themselves from positive feedback loops linked to the constant activation of the DNA damage response. This capacity requires changes in core cellular programs. Understanding how different cell types can undergo extensive changes in their transcriptional programs, metabolism, heterochromatin patterns, and cellular structures to induce a common cellular state is crucial to preventing cancer development/progression and to improving health during aging. In this review, we discuss how senescent cells continuously evolve after their initial proliferative arrest and highlight the unifying features that define the senescent state.

Eroded human telomeres are more prone to remain uncapped and to trigger a G2 checkpoint response.

Telomeres cap the ends of chromosomes and regulate the replicative life span of human somatic cells. Telomere function is lost upon critical shortening and a p53-dependent checkpoint that detects altered telomere states at the G1/S transition was proposed to act as a regulator of the telomere damage response. We show that telomerase-negative human fibroblasts spend more time in G2 phase as they approach senescence and this delay is associated with manifestations of telomere dysfunction and the triggering of an ATM/ATR-dependent DNA damage signal. This correlates with a partial release of telomeric proteins TRF1 and TRF2. Analysis of the consequences of TRF1 and TRF2 depletion or over-expression of mutated versions revealed that telomere uncapping or telomere replication stress also led to DNA damage signalling in G2. Progression through mitosis of these cells was associated with signs of incomplete telomere terminal processing. We also observed an increase in sister chromatid-type telomere aberrations in senescing fibroblasts indicating that defects of telomere post-replicative events increased as cells age. Our results link a post-replicative damage response at eroded telomeres to G2 arrest signalling and challenge the current paradigm that the checkpoint response to short telomeres occurs primarily at the G1/S transition in human cells.

Gain of oncogenic function of p53 mutants regulates E-cadherin expression uncoupled from cell invasion in colon cancer cells.

Mutations in the p53 tumour suppressor gene are associated clinically with tumour progression and metastasis. Downregulation of the E-cadherin cell-cell adhesion molecule is a key event for epithelial to mesenchymal transition (EMT) in tumour progression. Here, we show that wild-type p53 induced to adopt a mutant conformation, and hot-spot p53 mutants, which are both transcriptionally inactive, downregulate E-cadherin expression in the colon carcinoma cell line HCT116. Downregulation of E-cadherin occurred concomitantly with the upregulation of Slug and Zeb-1, transcriptional factors known to repress E-cadherin gene expression. In addition, knockdown of Slug and Zeb-1 expression diminished p53-mediated E-cadherin repression. Knocking down endogenous mutant p53 in MDA-MB-231 and SW620 cancer cell lines lacking E-cadherin protein restored the expression of E-cadherin. Complete loss of E-cadherin expression in HCT116 cells induced morphological alterations along with upregulation of vimentin, a mesenchymal marker. These changes characteristic of the EMT phenotype were, however, not sufficient to confer invasiveness in a three-dimensional matrix. Downregulation of E-cadherin by mutant p53 was not required to promote the invasive phenotype induced by inactivation of p53. These findings indicate that independent control of E-cadherin expression and cell motility could be essential molecular events in p53 mutant-induced invasive phenotypes.

p21-Mediated nuclear retention of cyclin B1-Cdk1 in response to genotoxic stress.

G2 arrest of cells suffering DNA damage in S phase is crucial to avoid their entry into mitosis, with the concomitant risks of oncogenic transformation. According to the current model, signals elicited by DNA damage prevent mitosis by inhibiting both activation and nuclear import of cyclin B1-Cdk1, a master mitotic regulator. We now show that normal human fibroblasts use additional mechanisms to block activation of cyclin B1-Cdk1. In these cells, exposure to nonrepairable DNA damage leads to nuclear accumulation of inactive cyclin B1-Cdk1 complexes. This nuclear retention, which strictly depends on association with endogenous p21, prevents activation of cyclin B1-Cdk1 by Cdc25 and Cdk-activating kinase as well as its recruitment to the centrosome. In p21-deficient normal human fibroblasts and immortal cell lines, cyclin B1 fails to accumulate in the nucleus and could be readily detected at the centrosome in response to DNA damage. Therefore, in normal cells, p21 exerts a dual role in mediating DNA damage-induced cell cycle arrest and exit before mitosis. In addition to blocking pRb phosphorylation, p21 directly prevents mitosis by inactivating and maintaining the inactive state of mitotic cyclin-Cdk complexes. This, with subsequent degradation of mitotic cyclins, further contributes to the establishment of a permanent G2 arrest.

[Senescence: a telomeric limit to immortality or a cellular response to physiologic stresses?]. / La sénescence: une barrière télomérique à l'immortalité ou une réponse cellulaire aux stress physiologiques?

Cells entering a state of senescence undergo a irreversible cell cycle arrest, associated by a set of functional and morphological changes. Senescence occurs following telomeres shortening (replicative senescence) or exposure to other acute or chronic physiologic stress signals (a phenomenon termed stasis: stress or aberrant signaling-induced senescence). In this review, I discuss the pathways of cellular senescence, the mechanisms involved and the role that these pathways have in regulating the initiation and progression of cancer. Telomere-initiated senescence or loss of telomere function trigger focal recruitement of protein sensors of the DNA double-strand breaks leading to the activation of the DNA damage checkpoint responses and the tumour suppressor gene product, p53, which in turn induces the cell-cycle inhibitor, p21(WAF1). Loss of p53 and pRb function allows continued cell division despite increasing telomere dysfunction and eventually entry into telomere crisis. Immortalisation is an essential prerequisite for the formation of a tumour cell. Therefore, a developing tumour cell must circumvent at least two proliferative barriers--cellular senescence and crisis--to achieve neoplastic transformation. These barriers are regulated by telomere shortening and by the p16(INK4a)/Rb and p53 tumour suppressor pathways. Elucidation of the genes and emerging knowledge about the regulatory mechanisms that lead to senescence and determine the pattern of gene expression in senescent cells may lead to more effective treatments for cancer.

Senescence from G2 arrest, revisited.

Senescence was classically defined as an irreversible cell cycle arrest in G1 phase (G1 exit) triggered by eroded telomeres in aged primary cells. The molecular basis of this G1 arrest is thought to be due to a DNA damage response, resulting in accumulation of the cyclin dependent kinase (Cdk) inhibitors p21 and p16 that block the inactivating phosphorylation of the retinoblastoma tumor suppressor pRb, thereby preventing DNA replication. More than a decade ago, several studies showed that p21 also mediates permanent DNA damage-induced cell cycle arrest in G2 (G2 exit) by inhibiting mitotic Cdk complexes and pRb phosphorylation. The idea that the senescence program can also be launched after G2 arrest has gained support from several recent publications, including evidence for its existence in vivo.

Greatwall promotes cell transformation by hyperactivating AKT in human malignancies.

The PP2A phosphatase is often inactivated in cancer and is considered as a tumour suppressor. A new pathway controlling PP2A activity in mitosis has been recently described. This pathway includes the Greatwall (GWL) kinase and its substrates endosulfines. At mitotic entry, GWL is activated and phosphorylates endosulfines that then bind and inhibit PP2A. We analysed whether GWL overexpression could participate in cancer development. We show that GWL overexpression promotes cell transformation and increases invasive capacities of cells through hyperphosphorylation of the oncogenic kinase AKT. Interestingly, AKT hyperphosphorylation induced by GWL is independent of endosulfines. Rather, GWL induces GSK3 kinase dephosphorylation in its inhibitory sites and subsequent SCF-dependent degradation of the PHLPP phosphatase responsible for AKT dephosphorylation. In line with its oncogenic activity, we find that GWL is often overexpressed in human colorectal tumoral tissues. Thus, GWL is a human oncoprotein that promotes the hyperactivation of AKT via the degradation of its phosphatase, PHLPP, in human malignancies.

The p53 isoform Δ133p53ß promotes cancer stem cell potential.

Cancer stem cells (CSC) are responsible for cancer chemoresistance and metastasis formation. Here we report that Δ133p53ß, a TP53 splice variant, enhanced cancer cell stemness in MCF-7 breast cancer cells, while its depletion reduced it. Δ133p53ß stimulated the expression of the key pluripotency factors SOX2, OCT3/4, and NANOG. Similarly, in highly metastatic breast cancer cells, aggressiveness was coupled with enhanced CSC potential and Δ133p53ß expression. Like in MCF-7 cells, SOX2, OCT3/4, and NANOG expression were positively regulated by Δ133p53ß in these cells. Finally, treatment of MCF-7 cells with etoposide, a cytotoxic anti-cancer drug, increased CSC formation and SOX2, OCT3/4, and NANOG expression via Δ133p53, thus potentially increasing the risk of cancer recurrence. Our findings show that Δ133p53ß supports CSC potential. Moreover, they indicate that the TP53 gene, which is considered a major tumor suppressor gene, also acts as an oncogene via the Δ133p53ß isoform.

Dysfunctional telomeres at senescence signal cell cycle arrest via Chk2.

Loss of telomere integrity can have two outcomes with opposite predicted effects on tumorigenesis. On the one hand, shortened telomeres in normal cells may trigger cell cycle arrest, leading to tumor suppression. On the other hand, in a tumor cell in which neither the p53 nor pRb pathway is intact, shortened telomeres could initiate chromosome instability and promote tumorigenesis A major issue in telomere research is to understand how shortened dysfunctional telomeres can regulate the onset of cellular senescence. Recent studies have revealed that critically shortened or acutely uncapped telomeres share molecular features with damaged DNA. We have recently linked the phosphorylation and activation of one major DNA damage effector checkpoint kinase, Chk2, to telomere erosion in signalling cell cycle arrest in normal fibroblasts. Here, we discuss several hypotheses to explain the molecular events occurring at shortened telomeres that ultimately lead to cell cycle arrest or increased genomic instability.

TNFalpha induces sequential activation of Cdc42- and p38/p53-dependent pathways that antagonistically regulate filopodia formation.

Cell migration is an essential function in various physiological processes, including tissue repair and tumour invasion. Repair of tissue damage requires the recruitment of fibroblasts to sites of tissue injury, which is mediated in part by the cytokine tumour necrosis factor alpha (TNFalpha). As dynamic rearrangements of actin cytoskeleton control cell locomotion, this implicates that TNFalpha is a potent coordinator of cellular actin changes. We have investigated the role of TNFalpha in regulating the cortical actin-containing structures essential for cell locomotion called filopodia. Kinetic analysis of TNFalpha-treated mouse embryonic fibroblasts (MEFs) revealed a dual effect on filopodia formation: a rapid and transient induction mediated by Cdc42 GTPase that is then counteracted by a subsequent sustained inhibition requiring activation of the mitogen-activated protein kinase p38 but not Cdc42 activity. This inhibition also involves the tumour suppressor p53, given that it is activated in response to TNFalpha following the same time course as the decrease of filopodia formation. This functional activation of p53, measured by transcription induction of its target p21WAF1(p21), is also associated with p38 kinase-dependent phosphorylation of p53 at serine 18. Furthermore, TNFalpha did not inhibit filopodia formation in MEFs treated with the transcription inhibitor actinomycin D, in p53-deficient MEFs, or MEFs expressing p53 mutants H273 or H175, which supports a role for the transcriptional activity of p53 in mediating TNFalpha-dependent filopodia inhibition. Our data delineate a novel inhibitory pathway in which TNFalpha prevents filopodia formation and cell migration through the activation of the mitogen-activated protein kinase (MAPK) p38, which in turn activates p53. This shows that TNFalpha on its own initiates antagonistic signals that modulate events linked to cell migration.

DNA damage checkpoint kinase Chk2 triggers replicative senescence.

Telomere shortening in normal human cells causes replicative senescence, a p53-dependent growth arrest state, which is thought to represent an innate defence against tumour progression. However, although it has been postulated that critical telomere loss generates a 'DNA damage' signal, the signalling pathway(s) that alerts cells to short dysfunctional telomeres remains only partially defined. We show that senescence in human fibroblasts is associated with focal accumulation of gamma-H2AX and phosphorylation of Chk2, known mediators of the ataxia-telangiectasia mutated regulated signalling pathway activated by DNA double-strand breaks. Both these responses increased in cells grown beyond senescence through inactivation of p53 and pRb, indicating that they are driven by continued cell division and not a consequence of senescence. gamma-H2AX (though not Chk2) was shown to associate directly with telomeric DNA. Furthermore, inactivation of Chk2 in human fibroblasts led to a fall in p21(waf1) expression and an extension of proliferative lifespan, consistent with failure to activate p53. Thus, Chk2 forms an essential component of a common pathway signalling cell cycle arrest in response to both telomere erosion and DNA damage.

Permanent cell cycle exit in G2 phase after DNA damage in normal human fibroblasts.

Although the Cdk inhibitor p21(Waf1/Cip1), one of the transcriptional targets of p53, has been implicated in the maintenance of G(2) arrest after DNA damage, its function at this stage of the cell cycle is not really understood. Here, we show that the exposure of normal human fibroblasts (NHFs) to genotoxic agents provokes permanent cell cycle exit in G(2) phase, whereas mouse embryo fibroblasts and transformed human cells progress through mitosis and arrest in G(1) without intervening cytokinesis. p21(Waf1/Cip1) exerts a key role in driving this G(2) exit both by inhibiting cyclin B1-Cdk1 and cyclin A-Cdk1/2 complexes, which control G(2)/M progression, and by blocking the phosphorylation of pRb family proteins. NHFs with compromised pRb proteins could still efficiently arrest in G(2) but were unable to exit the cell cycle, resulting in cell death. Our experiments show that, when under continuous genotoxic stress, normal cells can reverse their commitment to mitotic progression due to passage through the restriction point and that mechanisms involving p21(Waf1/Cip1) and pocket proteins can induce exit in G(2) and G(1).