Collaborator of ARF (CARF) Regulates Proliferative Fate of Human Cells by Dose-dependent Regulation of DNA Damage Signaling

Collaborator of ARF (CARF) has been shown to directly bind to and regulate p53, a central protein that controls tumor suppression via cellular senescence and apoptosis. However, the cellular functions of CARF and the mechanisms governing its effect on senescence, apoptosis, or proliferation are still unknown. Our previous studies have shown that (i) CARF is up-regulated during replicative and stress-induced senescence, and its exogenous overexpression caused senescence-like growth arrest of cells, and (ii) suppression of CARF induces aneuploidy, DNA damage, and mitotic catastrophe, resulting in apoptosis via the ATR/CHK1 pathway. In the present study, we dissected the cellular role of CARF by investigating the molecular pathways triggered by its overexpression in vitro and in vivo. We found that the dosage of CARF is a critical factor in determining the proliferation potential of cancer cells. Most surprisingly, although a moderate level of CARF overexpression induced senescence, a very high level of CARF resulted in increased cell proliferation. We demonstrate that the level of CARF is crucial for DNA damage and checkpoint response of cells through ATM/CHK1/CHK2, p53, and ERK pathways that in turn determine the proliferative fate of cancer cells toward growth arrest or proproliferative and malignant phenotypes. To the best of our knowledge, this is the first report that demonstrates the capability of a fundamental protein, CARF, in controlling cell proliferation in two opposite directions and hence may play a key role in tumor biology and cancer therapeutics.     

Anti-cancer effect of marchantin C via inducing lung cancer cellular senescence associated with less secretory phenotype

BackgroundLung cancer is the leading cause of global cancer deaths. Current chemotherapeutic agents for lung cancer treatment are generally accompanied with severe side effects. Here, we report that marchantin C (Mar-C), a potential natural compound with little chemotherapeutic toxicity, exerts a well anti-tumor effect against lung cancer via inducing cellular senescence.MethodsThe antitumor activity of Mar-C was evaluated by MTT and colony formation in vitro cytotoxicity assays, and xenograft and homograft in vivo model. Antitumor mechanisms of Mar-C were investigated through SA-β-gal staining, Q-PCR, immunoblotting, immunofluorescence, protein array and siRNA knocking-down analysis.ResultsMar-C selectively induces senescence of lung cancer cells with limited cytotoxicity on normal or non-neoplastic cells. Mar-C-induced senescence was associated with the elevation of ROS and activation of DNA-damage, and largely dependent of prolonged p21^CIP1 accumulation. The senescence-associated secretory phenotype (SASP) induced by Mar-C was distinct from doxorubicin-induced. Furthermore, Mar-C exhibited an inhibitory activity on tumor growth with little toxicity in animal studies, and significantly prolonged the survival time of tumor-bearing mice than that of doxorubicin or vehicle treatments.ConclusionMar-C selectively inhibited tumor growth via the induction of cancer cell senescence and had little chemotherapeutic toxicity, suggesting the potential of Mar-C as a promising anticancer agent.General significanceThis study provided evidence to identify a novelty of Mar-C that exerted antitumor activity on lung cancer through induction of senescence with limited toxicity.     

Computational experiments reveal the efficacy of targeting CDK2 and CKIs for significantly lowering cellular senescence bar for potential cancer treatment

Lowering the threshold of cellular senescence, the process employed by cells to thwart abnormal cell proliferation, though inhibition of CDK2 or Skp2 (regulator of CDK inhibitors) has been recently suggested as a potential avenue for cancer treatment. In this study, we employ a published mathematical model of G1/S transition involving the DNA-damage signal transduction pathway to conduct carefully constructed computational experiments to highlight the effectiveness of manipulating cellular senescence in inhibiting damaged cell proliferation. We first demonstrate the suitability of the mathematical model to explore senescence by highlighting the overlap between senescence pathways and those involved in G1/S transition and DNA damage signal transduction. We then investigate the effect of CDK2 deficiency on senescence in healthy cells, followed by effectiveness of CDK2 deficiency in triggering senescence in DNA damaged cells. For this, we focus on the behaviour of CycE, whose peak response indicates G1/S transition, for several reduced CDK2 levels in healthy as well as two DNA-damage conditions to calculate the probability (β) or the percentage of CDK2 deficient cells passing G1/S checkpoint ((1 - β) indicates level of senescence). Results show that 50% CDK2 deficiency can cause senescence in all healthy cells in a fairly uniform cell population; whereas, most healthy cells (≈67%) in a heterogeneous population escape senescence. This finding is novel to our study. Under both low- and high-DNA damaged conditions, 50% CDK deficiency can cause 65% increase in senescence in a heterogeneous cell population. Furthermore, the model analyses the relationship between CDK2 and its CKIs (p21, p27) to help search for other effective ways to bring forward cellular senescence. Results show that the degradation rate of p21 and initial concentration of p27 are effective in lowering CDK2 levels to lower the senescence threshold. Specifically, CDK2 and p27 are the most effective in triggering senescence while p21 having a smaller influence. While receiving experimental support, these findings specify in detail the inhibitory effects of CKIs. However, simultaneous variation of CDK2 and CKIs produces a dramatic reduction of damage cells passing the G1/S with CDK2&p27 combination causing senescence in almost all damaged cells. This combined effect of CDK2&CKIs on senescence is a novel contribution in this study. A review of the crucial protein complexes revealed that the concentration of active CycE/CDK2-p that controls cell cycle arrest provides support for the above findings with CycE/CDK2-p undergoing the largest reduction (over 100%) under the combined CDK2&CKI conditions leading to the arrest of most of the damaged cells. Our study thus provides quantitative assessments for the previously published qualitative findings on senescence and highlights new avenues for bringing forward senescence bar.     

Shp2 signaling suppresses senescence in PyMT-induced mammary gland cancer in mice

In this study, we have used techniques from cell biology, biochemistry, and genetics to investigate the role of the tyrosine phosphatase Shp2 in tumor cells of MMTV-PyMT mouse mammary glands. Genetic ablation or pharmacological inhibition of Shp2 induces senescence, as determined by the activation of senescence-associated β-gal (SA-β-gal), cyclin-dependent kinase inhibitor 1B (p27), p53, and histone 3 trimethylated lysine 9 (H3K9me3). Senescence induction leads to the inhibition of self-renewal of tumor cells and blockage of tumor formation and growth. A signaling cascade was identified that acts downstream of Shp2 to counter senescence: Src, focal adhesion kinase, and Map kinase inhibit senescence by activating the expression of S-phase kinase-associated protein 2 (Skp2), Aurora kinase A (Aurka), and the Notch ligand Delta-like 1 (Dll1), which block p27 and p53. Remarkably, the expression of Shp2 and of selected target genes predicts human breast cancer outcome. We conclude that therapies, which rely on senescence induction by inhibiting Shp2 or controlling its target gene products, may be useful in blocking breast cancer.     

HdmX overexpression inhibits oncogene induced cellular senescence

Cellular senescence is an irreversible state of terminal growth arrest that requires functional p53. Acting to block tumor formation, induction of senescence has also been demonstrated to contribute to tumor clearance via the immune system following p53 reactivation.^{1, 2} The Hdm2-antagonist, Nutlin-3a, has been shown to reactivate p53 and induce a quiescent state in various cancer cell lines,^{3, 4} similar to the G1 arrest observed upon RNAi targeting of Hdm2 in MCF7 breast cancer.⁵ In the present study we show that HdmX, a negative regulator of p53, impacts the senescence pathway. Specifically, overexpression of HdmX blocks Ras mediated senescence in primary human fibroblasts. The interaction of HdmX with p53 and the re-localization of HdmX to the nucleus through Hdm2 association appear to be required for this activity. Furthermore, inhibiting HdmX in prostate adenocarcinoma cells expressing wild-type p53, mutant Ras and high levels of HdmX induced cellular senescence as measured by an increase in irreversible b-galactosidase staining. Together these results suggest that HdmX overexpression may contribute to tumor formation by blocking senescence and that targeting HdmX may represent an attractive anti-cancer therapeutic approach.     

Dexamethasone Reduces Sensitivity to Cisplatin by Blunting p53-Dependent Cellular Senescence in Non-Small Cell Lung Cancer

Introduction

Dexamethasone (DEX) co-treatment has proved beneficial in NSCLC patients, improving clinical symptoms by the reduction of side effects after chemotherapy. However, recent studies have shown that DEX could render cancer cells more insensitive to cytotoxic drug therapy, but it is not known whether DEX co-treatment could influence therapy-induced senescence (TIS), and unknown whether it is in a p53-dependent or p53-independent manner.

Methods

We examined in different human NSCLC cell lines and detected cellular senescence after cisplatin (DDP) treatment in the presence or absence of DEX. The in vivo effect of the combination of DEX and DDP was assessed by tumor growth experiments using human lung cancer cell lines growing as xenograft tumors in nude mice.

Results

Co-treatment with DEX during chemotherapy in NSCLC resulted in increased tumor cell viability and inhibition of TIS compared with DDP treated group. DEX co-treatment cells exhibited the decrease of DNA damage signaling pathway proteins, the lower expression of p53 and p21^CIP1, the lower cellular secretory program and down-regulation of NF-κB and its signaling cascade. DEX also significantly reduced DDP sensitivity in vivo.

Conclusions

Our results underscore that DEX reduces chemotherapy sensitivity by blunting therapy induced cellular senescence after chemotherapy in NSCLC, which may, at least in part, in a p53-dependent manner. These data therefore raise concerns about the widespread combined use of gluocorticoids (GCs) with antineoplastic drugs in the clinical management of cancer patients.     

Inhibition of the Unfolded Protein Response by metformin in renal proximal tubular epithelial cells

All-trans retinoic acid (ATRA) induces cellular senescence via up-regulation of p16 and p21; however, the action mechanism of ATRA is unknown. Here, we show that ATRA induces promoter hypomethylation of p16 and p21 via down-regulation of DNA methyltransferases 1, 3a, and 3b to facilitate binding of Ets1/2 to the p16 promoter and p53 to the p21 promoter, resulting in up-regulation of their expression and subsequent induction of cellular senescence in HepG2 cells. These effects were mediated by retinoic acid receptor β₂ whose promoter was also hypomethylated in the presence of ATRA. Therefore, ATRA can be considered as an epi-drug in cancer therapy.     

Inhibition of Mcl-1 promotes senescence in cancer cells: implications for preventing tumor growth and chemotherapy resistance

Although senescence in oncogenesis has been widely studied, little is known regarding the role of this process in chemotherapy resistance. Thus, from the standpoint of enhancing and improving cancer therapy, a better understanding of the molecular machinery involved in chemotherapy-related senescence is paramount. We show for the first time that Mcl-1, a Bcl-2 family member, plays an important role in preventing chemotherapy-induced senescence (CIS). Overexpression of Mcl-1 in p53⁺ cell lines inhibits CIS. Conversely, downregulation of Mcl-1 makes cells sensitive to CIS. Surprisingly, downregulation of Mcl-1 in p53⁻ cells restored CIS to similar levels as p53⁺ cells. In all cases where senescence can be induced, we observed increased p21 expression. Moreover, we show that the domain of Mcl-1 responsible for its antisenescent effects is distinct from that known to confer its antiapoptotic qualities. In vivo we observe that downregulation of Mcl-1 can almost retard tumor growth regardless of p53 status, while overexpression of Mcl-1 in p53⁺ cells conferred resistance to CIS and promoted tumor outgrowth. In summary, our data reveal that Mcl-1 can inhibit CIS in both a p53-dependent and -independent manner in vitro and in vivo and that this Mcl-1-mediated inhibition can enhance tumor growth in vivo.     

ZNF313 is a novel cell cycle activator with an E3 ligase activity inhibiting cellular senescence by destabilizing p21WAF1

ZNF313 encoding a zinc-binding protein is located at chromosome 20q13.13, which exhibits a frequent genomic amplification in multiple human cancers. However, the biological function of ZNF313 remains largely undefined. Here we report that ZNF313 is an ubiquitin E3 ligase that has a critical role in the regulation of cell cycle progression, differentiation and senescence. In this study, ZNF313 is initially identified as a XIAP-associated factor 1 (XAF1)-interacting protein, which upregulates the stability and proapoptotic effect of XAF1. Intriguingly, we found that ZNF313 activates cell cycle progression and suppresses cellular senescence through the RING domain-mediated degradation of p21^WAF1. ZNF313 ubiquitinates p21^WAF1 and also destabilizes p27^KIP1 and p57^KIP2, three members of the CDK-interacting protein (CIP)/kinase inhibitor protein (KIP) family of cyclin-dependent kinase inhibitors, whereas it does not affect the stability of the inhibitor of CDK (INK4) family members, such as p16^INK4A and p15^INK4B. ZNF313 expression is tightly controlled during the cell cycle and its elevation at the late G1 phase is crucial for the G1-to-S phase transition. ZNF313 is induced by mitogenic growth factors and its blockade profoundly delays cell cycle progression and accelerates p21^WAF1-mediated senescence. Both replicative and stress-induced senescence are accompanied with ZNF313 reduction. ZNF313 is downregulated during cellular differentiation process in vitro and in vivo, while it is commonly upregulated in many types of cancer cells. ZNF313 shows both the nuclear and cytoplasmic localization in epithelial cells of normal tissues, but exhibits an intense cytoplasmic distribution in carcinoma cells of tumor tissues. Collectively, ZNF313 is a novel E3 ligase for p21^WAF1, whose alteration might be implicated in the pathogenesis of several human diseases, including cancers.     

SIRT6 Depletion Suppresses Tumor Growth by Promoting Cellular Senescence Induced by DNA Damage in HCC

The role of Sirtuin 6 (SIRT6) as a tumor suppressor or oncogene in liver cancer remains controversial. Thus, we identified the specific role of SIRT6 in the progression of hepatocellular carcinoma (HCC). SIRT6 expression was significantly higher in HCC cell lines and HCC tissues from 138 patients than in an immortalized hepatocyte cell line, THLE-2 and non-tumor tissues, respectively. SIRT6 knockdown by shRNA suppressed the growth of HCC cells and inhibited HCC tumor growth in vivo. In addition, SIRT6 silencing significantly prevented the growth of HCC cell lines by inducing cellular senescence in the p16/Rb- and p53/p21-pathway independent manners. Microarray analysis revealed that the expression of genes involved in nucleosome assembly was apparently altered in SIRT6-depleted Hep3B cells. SIRT6 knockdown promoted G2/M phase arrest and downregulation of genes encoding histone variants associated with nucleosome assembly, which could be attributed to DNA damage. Taken together, our findings suggest that SIRT6 acts as a tumor promoter by preventing DNA damage and cellular senescence, indicating that SIRT6 represents a potential therapeutic target for the treatment of HCC.     

PRPF19 regulates p53-dependent cellular senescence by modulating alternative splicing of MDM4 mRNA

Alteration of RNA splicing is a hallmark of cellular senescence, which is associated with age-related disease and cancer development. However, the roles of splicing factors in cellular senescence are not fully understood. In this study, we identified the splicing factor PRPF19 as a critical regulator of cellular senescence in normal human diploid fibroblasts. PRPF19 was downregulated during replicative senescence, and PRPF19 knockdown prematurely induced senescence-like cell cycle arrest through the p53–p21 pathway. RNA-sequencing analysis revealed that PRPF19 knockdown caused a switch of the MDM4 splicing isoform from stable full-length MDM4-FL to unstable MDM4-S lacking exon 6. We also found that PRPF19 regulates MDM4 splicing by promoting the physical interaction of other splicing factors, PRPF3 and PRPF8, which are key components of the core spliceosome, U4/U6.U5 tri-snRNP. Given that MDM4 is a major negative regulator of p53, our findings imply that PRPF19 downregulation inhibits MDM4-mediated p53 inactivation, resulting in induction of cellular senescence. Thus, PRPF19 plays an important role in the induction of p53-dependent cellular senescence.     

pRb or its cousins: Who controls the family business?

Comment on: Bazarov A, et al. Cell Cycle 2012; 11:1008–1013More than 90% of human cancers are of epithelial origin. Cellular senescence of human mammary epithelial cells (HMECs) is an important barrier that protects cells from immortalization; the first step in breast cancer development.¹ Although induction of tumor suppressor p16 is not evident in some types of normal human fibroblasts undergoing senescence,² in cultured HMECs, senescence occurs by a robust p16 induction, and cells that acquire silencing of p16Ink4a locus eventually proliferate and undergo senescence again by telomere shortening in a p53-dependent manner.¹ Therefore, p16 induction is a critical barrier to immortalize HMECs in culture. p16 inhibits kinase activity of Cdk4/6-cyclinD complexes, which inactivate three pRb family proteins: pRb, p107 and p130. However, the relative contribution of these three pRb family proteins to HMEC senescence is not well understood.In a recent issue of Cell Cycle, Bazarov et al. examined the role of each pRb family protein in p16-mediated senescence in breast cancer cell lines and in HMECs (Fig. 1).³ They showed that knockdown of each of the three pRb family proteins individually did not abrogate senescence mediated by ectopically expressed p16 in the breast cancer cell lines MDA-MB-231 and MCF7. However, the senescence induced by ectopic p16 was abrogated if they introduced E7, which inactivates all three pRb family proteins. Their data suggest that two of pRb family proteins can compensate for the loss of each pRb family protein to induce p16-mediated senescence in these cancer cells. The remaining question is whether all three pRb family members play an additive role, and whether the inactivation of at least two members of the pRb family is required to overcome p16-induced senescence in breast cancer cells. On the other hand, they showed that abrogation of pRb, but not of p107 and/ or p130, attenuates senescence in HMECs, suggesting a non-redundant critical role of pRb in HMEC senescence. These data are consistent with a recent report demonstrating that pRb has a non-redundant role in repressing DNA replication during H-ras-induced senescence of human fibroblasts,⁴ and explain why pRb, but not p107 or p130, is frequently mutated in cancer. Interestingly, although abrogation of pRb is critical for HMECs escaping senescence, simultaneous depletion of pRb together with either p107, p130 or both accelerates bypass of senescence. This suggests that p107 and p130 help pRb to trigger/maintain HMEC senescence in culture and possibly in vivo. Although each pRb family protein preferentially binds to different members of the E2F family,⁵ the contribution of each E2F family protein in escaping p16-mediated senescence remains unclear. Therefore, it will be interesting to see whether the critical role of pRb, and a supportive role of p130 and p107, in p16-mediated HMEC senescence depend on how each pRb family protein interacts with an E2F family protein.Open in a separate windowFigure 1. Contribution of pRb family proteins to p16-mediated senescence in breast cancer cells and HMECs. Knockdown of each of the three pRb family proteins in breast cancer cells does not abrogate ectopic p16-induced senescence, suggesting that either two of pRb family proteins can compensate for the loss of each pRb family proteins or all three of pRb family proteins play an additive role in p16-mediated senescence in breast cancer cells. On the other hand, knockdown of pRb, but not of p107 or p130, abrogates HMEC senescence, suggesting a non-redundant critical role for pRb in senescence of HMECs. However, the knockdown of either p107 or p130, in conjunction with pRb depletion, abrogates HMEC senescence more efficiently than pRb knockdown alone. This suggests a supporting role for p107 and p130 in maintaining HMEC senescence.Bazarov et al. also showed that even aggressive p53-negative breast cancer cells undergo cellular senescence upon ectopic p16 expression. These results are quite encouraging from an epigenetic therapy point of view. Silencing of p16 often occurs in breast cancer cells via promoter methylation. During DNA replication, cells require new p16 promoter methylation to keep p16 silenced. The observations of Bazarov et al. suggest that we may be able to stop the growth of even aggressive p53-negative breast cancers in patients by inducing p16 expression in cancer cells using DNA methylation inhibitors. Back to the question of running family business: “it appears that pRb is still the boss, but in some cases, it may get a helping hand from his cousins- p107 and p130.”