Senescence-like Arrest Research Articles

Abstract 2036: Characterizing PARP inhibitor and next-generation anti-androgen therapy responses in advanced prostate cancer

Abstract PARP inhibitors (PARPi) have been shown to be effective in prostate cancer patients with certain DNA repair defects, thus ushering in the era of precision medicine for this disease. Despite this advance, barriers remain which impede further progress including an incomplete understanding of therapeutic response. Our previous and on-going work shows that PARP inhibition leads to both cell death and a more cytostatic, senescence-like arrest. We hypothesize that persistent cells may later promote progression and treatment resistance. Recent clinical trials such as PROpel and TALAPRO-2 have demonstrated success in combining PARPi’s with next-generation anti-androgen therapies (NGAT) including abiraterone and enzalutamide. Whether NGAT’s are synthetic lethal and cell death promoting with PARP inhibition is poorly understood. Additionally, given the evolving clinical landscape for prostate cancer, it is important to test whether prior NGAT exposure may preclude response to combination therapy. In this study, we sought to characterize these combinations to better understand tumor cell response to treatment and the placement of treatment within the overall therapeutic paradigm. We’ve previously used the C4-2B metastatic castration-resistant prostate cancer cell line as a model which responds well to PARP inhibition. Here, we additionally utilize the C4-2B-derived enzalutamide and abiraterone resistant lines MDVR and AbiR, respectively. The combination of olaparib with abiraterone (recently approved from results of PROpel) was significantly more efficacious than either monotherapy in all three models. Additionally, combination of talazoparib and enzalutamide (approved from results of TALAPRO-2) was also more effective than monotherapy. These data suggest that prior development of NGAT resistance does not prevent benefit from NGAT and PARPi combinations. Although both combinations were found to cause significantly less cell growth, cells appeared more cytostatic rather than apoptotic, suggesting NGAT’s do not induce significant synthetic lethality with PARPi’s. Our work suggests that combining a PARPi with an NGAT promotes a persistent cytostasis which may give rise to eventual tumor progression and the development of resistance. Future studies are directed at further characterizing combinations with PARPi’s and the development of novel strategies to enhance their efficacy. Citation Format: Love A. Moore, Bryan Correa Gonzalez, Allen C. Gao, Alan P. Lombard. Characterizing PARP inhibitor and next-generation anti-androgen therapy responses in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2036.

Abstract A010: Using oncogenic pathway agonism to sensitize RAS-mutant cancers to immunotherapy

Abstract Overall, some 30% of human cancers are driven by mutant RAS proteins, which have proven relatively difficult to target. While immunotherapy is effective for NRAS-mutant melanoma, no options exist for resistant disease, and mutant KRAS-driven lung and pancreatic carcinomas are much less responsive overall. Oncogenic pathways are often key targets for inhibition. However, pathway agonism has not been systematically explored as a therapeutic approach. BRAF-mutant melanomas respond to BRAF inhibitors (BRAFi) due to decreased ERK signaling. They can recover signaling by acquiring activating RAS/MEK mutations, but exhibit decreased proliferation in the absence of inhibitor, suggesting that supraphysiologic ERK signaling also compromises fitness. In line with this, we observed that increasing ERK hyperactivation in RAS-mutant cancers, particularly in those with a high ERK activity, might elevate ERK signaling to a degree to induce senescence and also create an inflammatory tumor microenvironment. We have shown that a majority of RAS-mutant cancer cell lines we tested undergo senescence-like arrest following exposure to all FDA-approved BRAF inhibitors at clinically relevant doses. Simultaneous MEK or ERK inhibition allows the cells to recover proliferation, and a BRAFi which does not induce ERK activation fails to have any effect, confirming that ERK hyperactivation is the key driver of this response. Importantly, these findings generalize across NRAS-mutant and KRAS-mutant cancer cell lines including in fresh human pancreatic adenocarcinoma explants. Finally, our data in two novel genomically-characterized, immunocompetent models of NRAS-mutant melanoma and KRAS-mutant pancreatic adenocarcinoma support our in-vitro findings. Both RAS-mutant murine models demonstrate that anti-PD1 therapy is substantially more efficacious when mice are additionally treated with BRAFi. Molecular profiling of the tumor microenvironment reveals the expression of cytokines related to senescence and marked infiltration of activated CD8+ T-cells. In order to implement more potentially effective immunotherapies, we have analyzed the expression of different immune-checkpoint molecules and we have observed a high expression of LAG3 and TIM3 in the melanoma NRAS-mutant model. We will further analyze the effect of those checkpoints in combination with BRAFi to potentiate the anti-tumoral responses and tumor regression. We propose that oncogenic pathway agonism is a novel, effective, and untested strategy to induce proliferation arrest and sensitization to immunotherapy. The impact of this data is in the ability to broadly sensitize RAS-mutant cancers to immunotherapy in the settings of de-novo and acquired resistance. The direct connection between a tumor cell signaling vulnerability to immunotherapy response is a major point of novelty and provides a clear molecular rationale for pursuing combined targeted and immune-based therapy. Citation Format: Alvaro de Mingo Pulido, Charles Adelmann, Brittney Sell, Karol Prieto, Kimberly Nguyen, Cynthia Dixey, Ivannie Ortiz-Rivera, Marco Napoli, Elsa Flores, Jason Fleming, Karen Mann, Christin Burd, Kenneth Y. Tsai. Using oncogenic pathway agonism to sensitize RAS-mutant cancers to immunotherapy [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A010.

Single cell analysis reveals stochastic cell-to-cell variation in stress response and senescence program

Prior to the pandemic, public health experts argued that loneliness was among the most significant threats facing women’s health and wellbeing. As the COVID-19 pandemic brought our social lives to an abrupt pause in March, 2020, older adults were encouraged to remain isolated from friends and family. Social distancing guidelines led many older people to decrease social interactions with others. Using a community-based longitudinal study of women age 60+, we examined how changes in feelings of social connections with others influenced loneliness in October 2020 relative to prior to the pandemic (in September 2018). Our previous research has shown that psychological resilience decreases the negative consequences of major life stressors in later life. We hypothesized that women with high social consequences of the pandemic would experience increased loneliness, but resilience would buffer these effects. In line with our hypotheses, results showed that those who reported significant declines in social connectedness with others during the pandemic (i.e., high social consequences) experienced significant increases in loneliness (beta=0.125; p<0.001). Resilience, alternatively, was significantly associated with decreased loneliness (beta=-0.05; p<0.05), and buffered the social consequences of the pandemic. That is, as resilience increased, the social consequences of COVID-19 significantly declined (p<0.01), and resilience attenuated the negative consequences of high levels of social consequences of COVID-19 on loneliness, while those with high social consequences and low resilience experienced significant increases in loneliness in association with the pandemic. Based on our findings, we discuss potential clinical implications for resilience-based interventions for older adults.

Tumor Cell Escape from Therapy-Induced Senescence as a Model of Disease Recurrence after Dormancy.

Senescence, a durable form of growth arrest, represents a primary response to numerous anticancer therapies. Although the paradigm that senescence is "irreversible" has largely withstood the findings of tumor cell recovery from what has been termed "pseudo-senescence" or "senescence-like arrest," a review of the literature suggests that therapy-induced senescence in tumor cells is not obligatorily a permanent cell fate. Consequently, we propose that senescence represents one avenue whereby tumor cells evade the direct cytotoxic impact of therapy, thereby allowing for prolonged survival in a dormant state, with the potential to recover self-renewal capacity and contribute to disease recurrence.

Inducing cellular senescence in vitro by using genetically encoded photosensitizers.

Cellular senescence, a form of cell cycle arrest, is one of the cellular responses to different types of exogenous and endogenous damage. The senescence phenotype can be induced in vitro by oncogene overexpression and/or DNA damage. Recently, we have reported a novel mechanism of cellular senescence induction by mild genotoxic stress. Specifically, we have shown that the formation of a small number of DNA lesions in normal and cancer cells during S phase leads to cellular senescence-like arrest within the same cell cycle. Here, based on this mechanism, we suggest an approach to remotely induce premature senescence in human cell cultures using short-term light irradiation. We used the genetically encoded photosensitizers, tandem KillerRed and miniSOG, targeted to chromatin by fusion to core histone H2B to induce moderate levels of DNA damage by light in S phase cells. We showed that the cells that express the H2B-fused photosensitizers acquire a senescence phenotype upon illumination with the appropriate light source. Furthermore, we demonstrated that both chromatin-targeted tandem KillerRed (produces O2−) and miniSOG (produces 1O2) induce single-stranded DNA breaks upon light illumination. Interestingly, miniSOG was also able to induce double-stranded DNA breaks.

Calpain 2 Regulates Akt-FoxO-p27Kip1 Protein Signaling Pathway in Mammary Carcinoma

Calpain 2 Regulates Akt-FoxO-p27Kip1 Protein Signaling Pathway in Mammary Carcinoma

Ubiquitin-specific proteases 7 and 11 modulate Polycomb regulation of the INK4a tumour suppressor

An important facet of transcriptional repression by Polycomb repressive complex 1 (PRC1) is the mono-ubiquitination of histone H2A by the combined action of the Posterior sex combs (Psc) and Sex combs extra (Sce) proteins. Here, we report that two ubiquitin-specific proteases, USP7 and USP11, co-purify with human PRC1-type complexes through direct interactions with the Psc orthologues MEL18 and BMI1, and with other PRC1 components. Ablation of either USP7 or USP11 in primary human fibroblasts results in de-repression of the INK4a tumour suppressor accompanied by loss of PRC1 binding at the locus and a senescence-like proliferative arrest. Mechanistically, USP7 and USP11 regulate the ubiquitination status of the Psc and Sce proteins themselves, thereby affecting their turnover and abundance. Our results point to a novel function for USPs in the regulation and function of Polycomb complexes.

Persistent p21 Expression after Nutlin-3a Removal Is Associated with Senescence-like Arrest in 4N Cells

Nutlin-3a is a preclinical drug that stabilizes p53 by blocking the interaction between p53 and MDM2. In our previous study, Nutlin-3a promoted a tetraploid G(1) arrest in two p53 wild-type cell lines (HCT116 and U2OS), and both cell lines underwent endoreduplication after Nutlin-3a removal. Endoreduplication gave rise to stable tetraploid clones resistant to therapy-induced apoptosis. Prior knowledge of whether cells are susceptible to Nutlin-induced endoreduplication and therapy resistance could help direct Nutlin-3a-based therapies. In the present study, Nutlin-3a promoted a tetraploid G(1) arrest in multiple p53 wild-type cell lines. However, some cell lines underwent endoreduplication to relatively high extents after Nutlin-3a removal whereas other cell lines did not. The resistance to endoreduplication observed in some cell lines was associated with a prolonged 4N arrest after Nutlin-3a removal. Knockdown of either p53 or p21 immediately after Nutlin-3a removal could drive endoreduplication in otherwise resistant 4N cells. Finally, 4N-arrested cells retained persistent p21 expression; expressed senescence-associated beta-galactosidase; displayed an enlarged, flattened phenotype; and underwent a proliferation block that lasted at least 2 weeks after Nutlin-3a removal. These findings demonstrate that transient Nutlin-3a treatment can promote an apparently permanent proliferative block in 4N cells of certain cell lines associated with persistent p21 expression and resistance to endoreduplication.

SsDNA fragments induce cell senescence by telomere uncapping

Telomere uncapping is known to induce senescence by activating a DNA damage response (DDR). However, it is still unclear what structural features of uncapped telomeres activate DDR. One hypothesis is that the exposure of the telomeric single-stranded G-rich 3′ overhang triggers a DNA damage response and is, thus, equivalent to telomere uncapping. To mimic this, we compared the effects of two short single-stranded oligonucleotides, (TTAGGG) 2 and (CCCTAA) 2. G-rich oligonucleotides induced DNA damage foci containing γH2AX and senescence-like arrest, whilst C-rich oligonucleotides had no effect. Oligonucleotides did not co-localize with γΗ2ΑX foci, instead the induced DNA damage foci were preferentially localized at telomeres. BrdU incorporation assays showed that the effect of G oligonucleotides on γH2AX foci formation was cell cycle-dependent; entry of cells into S phase was necessary for subsequent DNA damage foci formation. Together, our results show that short G-rich single-stranded oligonucleotides induce telomere uncapping in a cell cycle-dependent manner, probably by titrating essential factors like Pot1 away from telomeres.

Accelerated senescence: An emerging role in tumor cell response to chemotherapy and radiation

Treatment of malignancies with chemotherapeutic drugs and/or radiotherapy is designed to eliminate the disease by depriving the tumor cell of its reproductive potential. Frequently, the desired effect of cell killing is achieved through the promotion of apoptosis; however, accumulating evidence suggests that apoptosis may not be the exclusive or even primary mechanism whereby tumor cells lose their self-renewal capacity after radiation or drug treatment, particularly in the case of solid tumors. While failure to undergo apoptosis in response to chemotherapeutic drugs or radiation may represent a mechanism of drug and radiation resistance, particularly in the case of leukemias and lymphomas, it is gradually being recognized that in the case of solid tumors, loss of reproductive capacity can occur through alternative pathways including reproductive cell death or mitotic catastrophe, through autophagic cell death, and as described below, through a terminally arrested state similar to replicative senescence. Studies building upon the phenomenon of replicative senescence in normal cells approaching the limit of their reproductive potential have identified a comparable senescence-like arrest as a component of the tumor cell response to chemotherapeutic drugs and radiation. This response, which has been termed “premature senescence”, “senescence-like growth arrest”, “stress-induced premature senescence”, and “accelerated senescence”, can also result from supraphysiological mitogenic signaling, sub-optimal culture conditions, and ectopic expression of oncogenes. Here, we will use the term “accelerated senescence” in our consideration of the morphological, biochemical, and molecular aspects of treatment-induced senescence, its relationship to classical replicative senescence, its prevalence in clinical specimens and the implications of accelerated senescence for the outcome of cancer therapy.

P53 Mediates Senescence-Like Arrest Induced by Chronic Replicational Stress

Previous studies have shown that exposure of cells to high levels of replicational stress leads to permanent proliferation arrest that does not require p53. We have examined cellular responses to therapeutically relevant low levels of replicational stress that allow limited proliferation. Chronic exposure to low concentrations of hydroxyurea, aphidicolin, or etoposide induced irreversible cell cycle arrest after several population doublings. Inhibition of p53 activity antagonized this arrest and enhanced the long-term proliferation of p53 mutant cells. p21CIP1 was found to be a critical p53 target for arrest induced by hydroxyurea or aphidicolin, but not etoposide, as judged by the ability of p21CIP1 suppression to mimic the effects of p53 disruption. Suppression of Rad51 expression, required for homologous recombination repair, blocked the ability of mutant p53 to antagonize arrest induced by etoposide, but not aphidicolin. Thus, the ability of mutant p53 to prevent arrest induced by replicational stress per se is primarily dependent on preventing p21CIP1 up-regulation. However, when replication stress is associated with DNA strand breaks (such as with etoposide), up-regulation of homologous recombination repair in response to p53 disruption becomes important. Since replicational stress leads to clonal selection of cells with p53 mutations, our results highlight the potential importance of chronic replicational stress in promoting cancer development.

Inhibitory role of RhoA on senescence-like growth arrest by a mechanism involving modulation of phosphatase activity

Recently, negative effects of phosphatase in tumorigenesis and metastasis have been suggested in various tumor types. In this study, we showed that RhoA activation modulated phosphatase during senescence-like arrest in human prostate cancer cells. Under senescence-inducing condition, decreased Erk phosphorylation was detected in caRhoA-transfected cells and inactivation of Erk, but not p38, prevented doxorubicin-induced cell senescence. Cells were induced to senescence by inhibition of phosphatase activity (VHR, MKP3, or PP2A) without additional cellular stress. Of interest, caRhoA prevented doxorubicin-induced decrease of phosphatase. Thus, we postulate that RhoA signaling may protect cells against cellular senescence by maintaining phosphatase activity and Erk dephosphorylation.

Mouse bites dogma: how mouse models are changing our views of how P53 is regulated in vivo

P53 is a transcription factor that can cause cells to be eliminated by apoptosis or senescent-like arrest upon its activation by irreparable genetic damage, excessively expressed oncogenes, or a broad spectrum of other stresses. As P53 executes life and death decisions, its activity must be stringently regulated, which implies that it is not likely to be controlled by a simple regulatory mechanism involving a binary on-off switch. This brief review will summarize a subset of the new information presented at the 10th P53 workshop in Dunedin, New Zealand in November 2004 as well as very recent publications that provide new insights into the molecular regulators of P53. Data emerging from mouse models provide a fundamentally different view of how P53 is regulated than suggested by more traditional in vitro approaches. The differences between cell culture and mouse models demonstrate the importance of preserving stoichiometric relationships between P53 and its various regulators to obtain an accurate view of the relevant molecular mechanisms that control P53 activity.

Akt negatively regulates the in vitro lifespan of human endothelial cells via a p53/p21-dependent pathway.

The signaling pathway of insulin/insulin-like growth factor-1/phosphatidylinositol-3 kinase/Akt is known to regulate longevity as well as resistance to oxidative stress in the nematode Caenorhabditis elegans. This regulatory process involves the activity of DAF-16, a forkhead transcription factor. Although reduction-of-function mutations in components of this pathway have been shown to extend the lifespan in organisms ranging from yeast to mice, activation of Akt has been reported to promote proliferation and survival of mammalian cells. Here we show that Akt activity increases along with cellular senescence and that inhibition of Akt extends the lifespan of primary cultured human endothelial cells. Constitutive activation of Akt promotes senescence-like arrest of cell growth via a p53/p21-dependent pathway, and inhibition of forkhead transcription factor FOXO3a by Akt is essential for this growth arrest to occur. FOXO3a influences p53 activity by regulating the level of reactive oxygen species. These findings reveal a novel role of Akt in regulating the cellular lifespan and suggest that the mechanism of longevity is conserved in primary cultured human cells and that Akt-induced senescence may be involved in vascular pathophysiology.

N-Methyl-N'-nitro-N-nitrosoguanidine-induced senescence-like growth arrest in colon cancer cells is associated with loss of adenomatous polyposis coli protein, microtubule organization, and telomeric DNA

BackgroundCellular senescence is a state in which mammalian cells enter into an irreversible growth arrest and altered biological functions. The senescence response in mammalian cells can be elicited by DNA-damaging agents. In the present study we report that the DNA-damaging agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is able to induce senescence in the HCT-116 colon cancer cell line.ResultsCells treated with lower concentrations of MNNG (0–25 microM) for 50 h showed a dose-dependent increase in G2/M phase arrest and apoptosis; however, cells treated with higher concentrations of MNNG (50–100 microM) showed a senescence-like G0/G1 phase arrest which was confirmed by increased expression of β-galactosidase, a senescence induced marker. The G2/M phase arrest and apoptosis were found to be associated with increased levels of p53 protein, but the senescence-like G0/G1 phase arrest was dissociated with p53 protein levels, since the p53 protein levels decreased in senescence-like arrested cells. We further, determined whether the decreased level of p53 was a transcriptional or a translational phenomenon. The results revealed that the decreased level of p53 protein in senescence-like arrested cells was a transcriptional phenomenon since p53 mRNA levels simultaneously decreased after treatment with higher concentrations of MNNG. We also examined the effect of MNNG treatment on other cell cycle-related proteins such as p21, p27, cyclin B1, Cdc2, c-Myc and max. The expression levels of these proteins were increased in cells treated with lower concentrations of MNNG, which supported the G2/M phase arrest. However, cells treated with higher concentrations of MNNG showed decreased levels of these proteins, and hence, may not play a role in cell cycle arrest. We then examined a possible association of the expression of APC protein and telomeric DNA signals with cellular senescence in MNNG-treated cells. We found that protein and mRNA levels of APC were drastically reduced in cells treated with higher concentrations of MNNG. The loss of APC expression might lead to chromosomal instability as well as microtubular disorganization through its dissociation with tubulin. In fact, the protein level of α-tubulin was also drastically decreased in senescence-like arrested cells treated with higher concentrations of MNNG. The levels of telomeric DNA also decreased in cells treated with higher concentrations of MNNG.ConclusionsThese results suggest that in response to DNA alkylation damage the senescence-like arrest of HCT-116 cells was associated with decreased levels of APC protein, microtubular organization, and telomeric DNA.

Extracellular Superoxide Dismutase Is a Major Antioxidant in Human Fibroblasts and Slows Telomere Shortening

There is good evidence that telomere shortening acts as a biological clock in human fibroblasts, limiting the number of population doublings a culture can achieve. Oxidative stress also limits the growth potential of human cells, and recent data show that the effect of mild oxidative stress is mediated by a stress-related increased rate of telomere shortening. Thus, fibroblast strains have donor-specific antioxidant defense, telomere shortening rate, and growth potential. We used low-density gene expression array analysis of fibroblast strains with different antioxidant potentials and telomere shortening rates to identify gene products responsible for these differences. Extracellular superoxide dismutase was identified as the strongest candidate, a correlation that was confirmed by Northern blotting. Over-expression of this gene in human fibroblasts with low antioxidant capacity increased total cellular superoxide dismutase activity, decreased the intracellular peroxide content, slowed the telomere shortening rate, and elongated the life span of these cells under normoxia and hyperoxia. These results identify extracellular superoxide dismutase as an important antioxidant gene product in human fibroblasts, confirm the causal role of oxidative stress for telomere shortening, and strongly suggest that the senescence-like arrest under mild oxidative stress is telomere-driven.

Iron chelation-induced senescence-like growth arrest in hepatocyte cell lines: association of transforming growth factor beta1 (TGF-beta1)-mediated p27Kip1 expression.

Iron is essential for cellular proliferation in all organisms. When deprived of iron, the growth of cells is invariably inhibited. However, the mechanism involved remains largely unclear. In the present study, we have observed that subcytotoxic concentrations of desferroxamine mesylate (DFO), an iron chelator, specifically inhibited the transition from G1 to S-phase of Chang cells, a hepatocyte cell line. This was accompanied by the appearance of senescent biomarkers, such as enlarged and flattened cell morphology, senescence-associated beta-galactosidase activity and reduced expression of poly(ADP-ribose) polymerase. Concomitantly, p27Kip1 (where Kip is kinase-inhibitory protein) was induced markedly, whereas other negative cell-cycle regulators, such as p21Cip1 (where Cip is cyclin-dependent kinase-interacting protein), p15INK4B and p16INK4A (where INK is inhibitors of cyclin-dependent kinase 4), were not, implying its association in the G1 arrest. Furthermore, the induction of p27Kip1 was accompanied by an increased level of transforming growth factor beta1 (TGF-beta1) mRNA. When neutralized with an anti-(TGF-beta1) antibody, p27Kip1 induction was completely abolished, indicating that TGF-beta1 is the major inducer of p27Kip1. Finally, DFO-induced senescence-like arrest was found to be independent of p53, since cell-cycle arrest was still observed with two p53-negative cell lines, Huh7 and Hep3B cells. In conclusion, DFO induced senescence-like G1 arrest in hepatocyte cell lines and this was associated with the induction of p27Kip1 through TGF-beta1, but was independent of p53.

Identification of Telomere-Dependent “Senescence-like” Arrest in Mouse Embryonic Fibroblasts

In contrast to human primary cells, mouse embryonic fibroblasts (MEF) do not show telomere shortening-mediated replicative senescence due to the fact that they have telomerase activity and show sufficiently long telomeres. Instead, it is now generally accepted that the “senescence-like” arrest that occurs in MEF after 5–10 divisions in culture is mediated by telomere-length-independent mechanisms generally referred to as stress. Using telomerase-deficient MEF Terc−/−, we show here that telomere shortening to a critical length leads to a premature senescence-like arrest in MEF, as well as has a negative effect on spontaneous immortalization. Similarly, elimination of the telomere end-capping protein Ku86 also leads to a premature senescence-like arrest and has a negative effect on spontaneous immortalization. Both Terc−/− MEF with short telomeres and Ku86−/− MEF show dysfunctional telomeres, as indicated by similarly increased frequencies of end-to-end fusions. These results suggest that loss of telomere function is a general mechanism leading to cell arrest. These observations also indicate that telomere dysfunction is interfering with successful cell division and thus interferes with tumor formation. In summary, we have identified here two different ways to induce a telomere-dependent senescence-like arrest in MEF.

Dual Growth Arrest Pathways in Astrocytes and Astrocytic Tumors in Response to Raf-1 Activation

Normal human fibroblasts have been shown to undergo a p16(Ink4a)-associated senescence-like growth arrest in response to sustained activation of the Ras/Raf/MEK/ERK pathway. We noted a similar p16(Ink4a)-associated, senescence-like arrest in normal human astrocytes in response to expression of a conditional form of Raf-1. While HPV16 E7-mediated functional inactivation of the p16(Ink4a)/pRb pathway in astrocytes blocked the p16(Ink4a)-associated growth arrest in response to activation of Raf-1, it also revealed a second p21(Cip1)-associated, senescence-associated, beta-galactosidase-independent growth arrest pathway. Importantly, the p21(Cip1)-associated pathway was present not only in normal astrocytes but also in p53-, p14(ARF)-, and p16(Ink4a)/pRb-deficient high grade glioma cells that lacked the p16(Ink4a)-dependent arrest mechanism. These results suggest that normal human cells have redundant arrest pathways, which can be activated by Raf-1, and that even tumors that have dismantled p16(Ink4a)-dependent growth arrest pathways are potentially regulated by a second p21(Cip1)-dependent growth arrest pathway.

Inhibition of the Phosphoinositide 3-Kinase Pathway Induces a Senescence-like Arrest Mediated by p27Kip1

A senescence-like growth arrest is induced in mouse primary embryo fibroblasts by inhibitors of phosphoinositide 3-kinase (PI3K). We observed that senescence-like growth arrest is correlated with an increase in p27(Kip1) but that down-regulation of other cyclin-dependent kinase (CDK) inhibitors, including p15(INK4b), p16(INK4a), p19( INK4d), and p21(Cip1) as well as other negative cell cycle regulators such as p53 and p19(ARF), implies that this senescence-related growth arrest is independent of the activity of p53, p19(ARF), p16(INK4a), and p21(Cip1), which are associated with replicative senescence. The p27(Kip1) binds to the cyclin/CDK2 complexes and causes a decrease in CDK2 kinase activity. We demonstrated that ectopic expression of p27(Kip1) can induce permanent cell cycle arrest and a senescence-like phenotype in wild-type mouse embryo fibroblasts. We also obtained results suggesting that the kinase inhibitors LY294002 and Wortmannin arrest cell growth and induce a senescence-like phenotype, at least partially, through inhibition of PI3K and protein kinase B/Akt, activation of the forkhead protein AFX, and up-regulation of p27(Kip1)expression. In summary, these observations taken together suggest that p27(Kip1) is an important mediator of the permanent cell cycle arrest induced by PI3K inhibitors. Our data suggest that repression of CDK2 activity by p27(Kip1) is required for the PI3K-induced senescence, yet mouse embryo fibroblasts derived from p27(Kip1-/-) mice entered cell cycle arrest after treatment with LY294002. We show that this is due to a compensatory mechanism by which p130 functionally substitutes for the loss of p27(Kip1). This is the first description that p130 may have a role in inhibiting CDK activity during senescence.