- Open Access
Temporally distinct roles for tumor suppressor pathways in cell cycle arrest and cellular senescence in Cyclin D1-driven tumor
© Zalzali et al.; licensee BioMed Central Ltd. 2012
- Received: 2 January 2012
- Accepted: 10 April 2012
- Published: 1 May 2012
Cellular senescence represents a tumor suppressive response to a variety of aberrant and oncogenic insults. We have previously described a transgenic mouse model of Cyclin D1-driven senescence in pineal cells that opposes tumor progression. We now attempted to define the molecular mechanisms leading to p53 activation in this model, and to identify effectors of Cyclin D1-induced senescence.
Senescence evolved over a period of weeks, with initial hyperproliferation followed by cell cycle arrest due to ROS production leading to activation of a DNA damage response and the p53 pathway. Interestingly, cell cycle exit was associated with repression of the Cyclin-dependent kinase Cdk2. This was followed days later by formation of heterochromatin foci correlating with RB protein hypophosphorylation. In the absence of the Cdk4-inhibitor p18Ink4c, cell cycle exit was delayed but most cells eventually showed a senescent phenotype. However, tumors later arose from this premalignant, largely senescent lesion. We found that the p53 pathway was intact in tumors arising in a p18Ink4c-/- background, indicating that the two genes represent distinct tumor suppressor pathways. Upon tumor progression, both p18Ink4c-/- and p53-/- tumors showed increased Cdk2 expression. Inhibition of Cdk2 in cultured pre-tumorigenic and tumor cells of both backgrounds resulted in decreased proliferation and evidence of senescence.
Our findings indicate that the p53 and the RB pathways play temporally distinct roles in senescence induction in Cyclin D1-expressing cells, and that Cdk2 inhibition plays a role in tumor suppression, and may be a useful therapeutic target.
- Cyclin D1
- Reactive oxygen species
Cellular senescence is a well-established tumor suppressor mechanism, activated in response to oncogenic signals, DNA damage, and telomere attrition among other pro-tumorigenic insults (reviewed in [1, 2]). Mechanistic insight into oncogene-induced senescence has emerged over the past few years. While it is clear that both the p53 and the Rb tumor suppressors are involved [3–6], their relative importance seems to vary depending on the activating insult and cellular context [3, 7–10]. Understanding the relative contributions of p53 and Rb to the induction and maintenance of senescence may have important implications, especially with development of targeted therapeutic agents.
Cyclin-dependent kinase 2 (Cdk2) was recently implicated in the senescence process, as Cdk2 loss was found to enhance senescence in Myc-induced tumors . In addition, it was shown that Cdk2-dependent phosphorylation of Myc was necessary to bypass Ras-induced senescence . This suggests that Cdk2 may act in senescence independently of its role in RB phosphorylation and cell cycle exit.
Here, we used a transgenic mouse model of premalignant Cyclin D1-driven pineal gland hyperplasia, to define the molecular mechanisms leading to p53 activation in response to Cyclin D1, and to identify effectors of Cyclin D1-induced senescence. The results shed light on the pattern of evolution of the senescence response in a premalignant lesion in vivo, and the differences in the contribution of the two major tumor suppressor pathways, p53 and RB. In addition, our findings suggest that Cdk2 inhibition may be a useful therapeutic approach, irrespective of the underlying genetic insult that led to senescence evasion.
We hypothesized that Cyclin D1 expression may be inducing p53 through activation of the DNA damage response (DDR), as reported for Ras-induced senescence (reviewed in ). Indeed, we found nuclear accumulation of phosphorylated histone H2AX (pH2AX) at P10, concomitant with p53 activation [Figure 2B, 2C]. In addition, Chk1 was phosphorylated concomitantly with phosphorylation of p53, further indicating that the DDR pathway was active in the transgenic pineal gland at this time, but not at later time-points [Figure 2D]. These findings reveal that deregulated Cyclin D1 enhances the DDR pathway and activates p53 while cells are proliferating, but ongoing DDR and active p53 are not needed after cells have undergone senescence.
To understand the mechanism of Rb activation, we investigated the expression of the Cdk-inhibitors p16Ink4a, p15Ink4b, p18Ink4c, and p27Kip1 [27–30]. There was increased expression of p18Ink4c at all time points [Figure 3B], an increase in p15Ink4b expression at P49 [see Figure 1D, but no changes in expression of p16Ink4a and p27Kip1 (not shown). We also evaluated the expression of Cdk4 and Cdk2, especially since Cdk2 inhibition was recently found to be important for Myc-induced senescence . We observed a modest decrease in Cdk4 expression from P10 through P49 [Figure 3B], but interestingly we found that Cdk2 expression was markedly reduced from P10 to P24 [Figure 3B], coincident with the timing of cell proliferation arrest and loss of Rb phosphorylation at Cdk2-specific sites. We conclude that Cdk2 repression correlates most closely with the initial proliferation arrest; and that diminished Cdk4-dependent Rb phosphorylation occurs at a later time-point and correlates with formation of SAHF.
p18 Ink4c loss delays p53-dependent cell cycle exit: Because of the observed increase in expression of p18Ink4c, we used a genetic approach to evaluate its role in Cyclin D1-induced senescence. In contrast to what occurs without p53 – where cell proliferation only slightly decreases from P10 to P35 and then increases as invasive tumor progresses [Figure 4C, top panel; and ], proliferation decreased from P10 through P49 without p18Ink4c [Figure 4C, bottom panel], but exceeded that in the Irbp-Cyclin D1 cells [Figure 4C, bottom panel, compare with Figure 1B].
Notably, p53 activation measured by phosphorylation at Ser15/20, and expression of the p53-target p21Cip1, persisted until P24 in Irbp-Cyclin D1, p18Ink4c -/- cells [Figure 4D], correlating with the prolonged cellular proliferation. Further, Cdk2 expression persisted until P24 in Irbp-Cyclin D1, p18Ink4c -/- cells [Figure 4]. These findings indicate that p18Ink4c loss delayed but did not prevent p53-dependent events leading to cell cycle exit.
Interestingly, loss of Cdk4-dependent Rb phosphorylation still occurred in the absence of p18Ink4c, again correlating with the appearance of SAHF [Figure 4D]. In fact, the majority of Irbp-Cyclin D1, p18Ink4c -/- cells displayed SAHF by P49 Additional file 2: Figure S2A], whereas SAHF never formed in Irbp-Cyclin D1, p53 -/- cells . In addition to SAHF, the senescence markers Dec1 and DcR2 were also expressed in Irbp-Cyclin D1, p18Ink4c -/- cells at P49 Additional file 1: Figure S2B]. Findings were similar in vitro using pineal cells explanted from P10 animals and cultivated for 10-20 days: Explanted Irbp-Cyclin D1 cells showed evidence of senescence, including loss of proliferation (measured by BrdU incorporation), and positive staining for SABG, by 10 days in culture Additional file 1: Figure S1B, 1C], while the Irbp-Cyclin D1, p53 -/- cells continued to proliferate and did not senesce Additional file 1: Figure S1B, bottom]. In contrast, the Irbp-Cyclin D1, p18Ink4c -/- cells did show evidence of senescence, but it was delayed until close to 20 days in culture Additional file 2: Figure S2C]. We conclude that p18Ink4c slowed proliferation but was not essential for most Cyclin D1 expressing cells to cease proliferating and become senescent.
We considered whether the prolonged proliferation in the absence of p18Ink4c might have derailed a p53-dependent arrest in the malignant tumors. Western blotting showed that Irbp-Cyclin D1, p18Ink4c -/- tumors still expressed the p53 protein [Figure 5D], and sequencing of p53 exons 5-8 did not reveal mutations in genomic DNA from nine different Irbp-Cyclin D1, p18Ink4c -/- pineal tumors (data not shown). Further, using primary cultures of pineal tumor cells, we found that both gamma irradiation and treatment with etoposide resulted in increased p53 phosphorylation and in p53-dependent increases in p21Cip1 and 14-3-3 in Irbp-Cyclin D1, p18Ink4c -/- but not Irbp-Cyclin D1, p53 -/- tumor cells [Figure 5E]. These findings confirmed that p53 remained intact in Irbp-Cyclin D1, p18Ink4c -/- tumor cells. In contrast, there was decreased p18Ink4c expression in Irbp-Cyclin D1, p53-/- tumors, suggesting that p18Ink4c may act as a tumor suppressor, even in a p53-null setting [Figure 5D, 5F]. However, preliminary results show no enhanced tumor susceptibility in Irbp-Cyclin D1, p53-/-, p18Ink4c -/- (double knock-out) animals (data not shown).
To address the role of Cdk2 repression as a possible therapeutic target, we treated explanted Irbp-Cyclin D1, p18Ink4c -/- and Irbp-Cyclin D1, p53 -/- pineal tumor cells with the Cdk2-inhibitor CVT313, at a concentration of 5 μM, known to specifically inhibit Cdk2 [11, 31]. CVT313 treatment decreased cell number (estimated by total cellular area) in Irbp-Cyclin D1, p18Ink4c -/- and Irbp-Cyclin D1, p53 -/- tumor cells in 8-well chamber slides [Figure 6D, upper panel]. Furthermore, CVT313-treated cells showed an increase in positive staining for SABG activity [Figure 6D, lower panel, and Figure 6E]. Importantly, treatment of pre-tumorigenic Irbp-Cyclin D1 pineal cells with CVT313 also decreased the apparent cell number [Figure 6E], while treatment of wild-type pineal cells did not seem to have a noticeable effect, either on cellularity or SABG positivity [Figure 6E].
To investigate whether the effects on senescence induction were specific to Cdk2 inhibition, we treated explanted Irbp-Cyclin D1, p53-/- and Irbp-Cyclin D1, p18Ink4c-/- cells with a specific Cdk4-inhibitor, NSC 625987 [32, 33]. Inhibition of Cdk4 decreased proliferation (measured by BrdU incorporation), though to a lesser extent than seen with Cdk2 inhibition [Figure 7C]. However, unlike Cdk2 inhibition, it did not result in any detectable increase in SABG staining [Figure 7D]. This demonstrates that Cdk2 inhibition was specifically relevant to induction of senescence in Cyclin D1-expressing pineal cells.
Emerging evidence supports the concept that cellular senescence represents a likely mechanism through which oncogenic transformation is suppressed. Senescence has been observed in pre-malignant lesions in mouse and in man, but not in fully transformed counterparts of these lesions (reviewed in [21, 34]). Because of the time lag in progression of premalignant lesions and the incomplete penetrance, it has been assumed that accumulation of as yet poorly-defined genetic or epigenetic changes likely contribute to the emergence of a tumor from a premalignant, apparently senescent lesion.
Our work provides new insight into cellular and molecular events that occur as oncogene-expressing cells arrest, become senescent, and finally emerge or escape from the senescent state as a malignant tumor. This is the first in vivo description of temporal morphologic and molecular events accompanying the evolution of an oncogene-driven senescent state, showing a previously unrecognized temporal sequence where cell cycle exit preceded formation of heterochromatin foci by several weeks.
Two tumor suppressor genes, p53 and p18Ink4c, played distinct roles during this process. P53 activation occurred concomitantly with an active DNA damage response, and was essential to drive cell cycle exit, temporally associated with Cdk2 repression and loss of Cdk2-dependent phosphorylation of the retinoblastoma protein Rb. Days later, reversal of Cdk4-dependent phosphorylation of the Rb protein correlated with the emergence of morphological and biochemical changes of oncogene-induced senescence. At that point, though, there was no evidence of p53 pathway activation. This is the first direct in vivo evidence for distinct temporal roles for these two tumor suppressors in the senescence process.
The early and transient activation of the p53 pathway suggested that p53 was integral for the initial cell cycle exit but not directly involved in formation of SAHF. Other models have also shown conflicting and context-dependent evidence for the role of p53 in the formation of SAHF [35–37]. In contrast, Rb activation was delayed and stable. Rb seemed to be important in both cell cycle exit as well as formation of SAHF: compromise of the Rb pathway through loss of p18Ink4c (the major known function of which is inhibition of Cdk4/6 to indirectly activate Rb [38–40]) led to a delay in initial cell cycle exit, and eventually to complete penetrance of tumor progression within the senescent-like lesion. Taken together, these findings implicate Rb, rather than p53, as the key protein needed to foster the emergence and maintenance of SAHF, thought to be responsible for repression of cell-cycle genes [3, 35]. Involvement of Rb in the formation of SAHF has been shown in other settings: human RB was shown to directly co-localize with SAHF [35, 36], and inactivation of the p16INK4a/RB pathway impairs formation of RasG12V-induced SAHF in human fibroblasts . In our model, p53-driven cell cycle exit correlated with hypo-phosphorylation of Rb at Cdk2-dependent sites, while formation of SAHF correlated with hypo-phosphorylation at Cdk4-dependent sites. This suggests that hypo-phosphorylation of these specific residues may be involved in SAHF formation. It will be interesting to evaluate whether mutated forms of Rb that cannot be phosphorylated at these particular Cdk4 sites can more robustly foster the appearance of SAHF.
Our results suggest that p53 and p18Ink4c represent separate tumor suppressor pathways in Cyclin D1-driven pineoblastoma. While tumors progressed within 3-4 months in p53 -/- animals, they appeared much later, after 7-10 months in p18Ink4c -/- mice. Also, the p53 pathway was intact in p18Ink4c -/- tumors, further proving that the two pathways of tumor suppression are distinct. Tumor suppression required functional p53 and p18Ink4c, as neither was sufficient to prevent tumor progression alone. While the cell cycle exit after P10 was clearly p53-dependent, absence of p18Ink4c delayed the cell cycle exit but did not prevent it in the majority of cells, which went on to express other markers of senescence. However, few cells continued to proliferate, resulting in tumorigenesis. It thus appears that, while p53 loss resulted in abrogation of cell cycle exit altogether, loss of p18Ink4c decreased the threshold for bypass of the p53-dependent cell cycle exit in a subset of cells.
In our model, p53-dependent cell cycle arrest was associated with marked Cdk2 repression, while Cdk2 levels were maintained in Irbp-Cyclin D1, p53 -/- cells which never exited the cell cycle [see Figure 5F. While a role for Cdk2 repression in facilitating senescence has been shown in an earlier report , ours is the first description of Cdk2 repression occurring in a temporal association with p53-dependent cell cycle exit. This indicates that Cdk2 repression might be a novel p53-dependent mechanism to foster cell cycle exit, especially since no similar changes were seen in the related cell cycle regulator Cdk1. However, additional work will be needed to investigate whether Cdk2 repression is a direct p53-dependent effect, and whether it is sufficient to induce cell cycle exit and induction of senescence. Moreover, since Cdk2 and other Cdks are also regulated post-transcriptionally by phosphorylation and by their binding to CDK-inhibitors, future work should focus on elucidating these molecular aspects for complete mechanistic understanding of the role of Cdk2 and other Cdks in inducing senescence. Future studies utilizing the established in vitro model, as well as genetically engineered mouse models, should be able to specifically dissect the role of Cdk2 in tumor progression, and upstream and downstream mechanisms leading to its repression and to cell cycle exit.
Finally, although tumors arising in a p53 -/- setting were molecularly different from those arising in a p18Ink4c -/- setting, Cdk2 levels were high in both (compared to senescent cells) and both cell types responded to Cdk2 inhibition. While Cdk4 inhibition also decreased cell proliferation, only Cdk2 inhibition resulted in features of senescence in treated cells. These conclusions are based on the published specificity of the inhibitors used at the corresponding concentrations. Ideally, we would have preferred to document inhibitor-induced pRb specific phosphorylations and specific kinase activity in each experiment, however the low number of primary cells used in these experiments was prohibitive.
Keeping the above limitations in mind, these findings provide a rationale for exploring the use of pharmacological Cdk inhibition, specifically Cdk2, to induce senescence in tumor cells, irrespective of whether the p53 pathway is compromised. Such an approach to therapy may be especially useful in tumors where the primary insult lies with deregulated Cyclin D1 expression, as in the reported model.
Irbp-Cyclin D1 transgenic mice  were bred with p53 -/- (Jackson Laboratory, Maine), or p18Ink4c -/- mice  and maintained in a mixed C57BL/6 × 129/Sv genetic background. PCR for targeted alleles was used to verify mouse genotypes as described [41, 42]. Animals were euthanized at defined time points or when obviously ill in accordance with the American University of Beirut (AUB) Institutional Animal Care and Use Committee guidelines; all studies were approved by this committee.
Analyses of protein expression
Protein lysates were prepared from pineal tissue by lysis in Universal Lysis Buffer. Electrophoresis was performed using 8, 10, or 12% Tris-Chloride gels, transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA), and detected using antibodies to p21Cip1, Cdk4, Cdk2, Cdk1, Hsc70, phospho-S790 Rb, total and phospho-specific p53 at Ser15/20 (Santa Cruz Biotechnology, Santa Cruz, CA); phospho-S612 Rb (MBL International, Woburn, MA); p18Ink4c (Zymed, San Francisco, CA); human Cyclin D1 (BD-Pharmingen, San Diego, CA); hemagglutinin (HA) epitope (Covance, Trenton, NJ); and 14-3-3 (Abcam, Cambridge, UK).
For evaluation of p53 pathway, pineal tumors were excised, dispersed, and plated onto permanox chamber slides, and grown in culture in 10% FBS in DMEM for 72 hours, then treated with 6 Gy irradiation at 1 Gy/min, or 10 μM Etoposide. Cells were collected 24 hours after irradiation, or 6 hours after Etoposide treatment, and protein extraction and western blotting was performed as above.
Histological studies and Immunostaining
Brain tissue was fixed in 4% paraformaldehyde for 72 hours then embedded in paraffin. For mice older than 10 days, skulls were peeled off before embedding. For BrdU incorporation, 49 day-old mice were treated with intraperitoneal injection of 50 mg/Kg of BrdU (Sigma-Aldrich, St Louis, MO), every 2 hours × 5, then sacrificed 2 hours later. 4-8 μm sections were cut from paraffin-embedded tissues and deparaffinized. Antigen retrieval was performed in a microwave at high power for 5 minutes, followed by low power for 5 minutes x2 in citrate antigen retrieval buffer (pH 6.0). Slides were incubated with anti-Ki67 (NovoCastra, Newcastle, UK), anti-pH2AX (Cell Signaling Technology, Danvers, MA), anti-Dec1, anti-DcR2, anti-MnSOD, anti-p15Ink4b, or anti-Cdk2 antibodies(Santa Cruz Biotechnology), followed by biotinylated secondary antibody; and detected using streptavidin conjugated to horseradish peroxidase and DAB substrate (DAKO, Carpinteria, CA). For immunofluorescence staining, anti-H3K9me3 (Upstate Laboratories, Syracuse, NY), anti-4HNE (Alpha Diagnostic, San Antonio, TX, USA), anti-BrdU, anti-p21 (Santa Cruz Biotechnology), anti-phosphorylated Chk1 at Ser345 (Cell Signaling Technology), anti-14-3-3, and anti-8(OH)dG (Abcam, Cambridge, UK) antibodies were detected with Cyanine 2, Cyanine 3, or Alexafluor488 secondary antibodies. The number of Ki67 positive cells, pH2AX positive cells, and BrdU-positive cells was manually counted from 5-7 representative fields, at 200x magnification, and normalized to total cell number. Digital photomicrographs were obtained using a Zeiss 510 NLO multiphoton/ confocal laser scanning microscope. Composite images were constructed using Photoshop CS4 software (Adobe Systems, Mountain View, CA).
Cell Explantation and ex-vivo culture
Pineal cells were explanted at postnatal day 10 (P10); tumors were explanted when clinically apparent (bulging cranium). Cells were plated onto 8-well permanox chamber slides (Nunc, Rochester, NY), and cultured in DMEM with 10%FBS, 1% glutamine, and 1% Pen/Strep.
To measure intracellular ROS in vitro, cells were treated with a peroxide sensitive reagent CM-H2DCF-DA (CM-H2DCF-DA; Molecular Probes, Eugene, OR) at 10 μM for 20 min at 37°C and observed under a fluorescence microscope.
N-Acetyl Cysteine treatment
Explanted pineal cells were treated with N-Acetyl Cysteine (Sigma Aldrich) at a concentration of 5 mM. Media was renewed daily. Cells were treated for 10 days, and stained for SABG as described . For DDR pathway analysis, cells were fixed and stained after 4 days.
CVT313 and NSC625987 treatment
Explanted cells were treated with CVT313 at 5 μM (Santa Cruz Biotechnology), NSC625987 at 1 μM (Tocris Bioscience, Ellisville, MO, USA), or DMSO vehicle; media was renewed every 3 days. Cells were fixed and stained for SABG after 7 days, and counterstained with eosin. For quantification of proportion of cells positive for SABG, 10 random fields were selected, and digital photomicrographs were analyzed using Adobe Photoshop CS4 software, by color selection and area analysis. For quantification of cellular accumulation, all the area of the well was photographed over 12 fields. Digital photomicrographs were analyzed using Adobe Photoshop CS4 software, by area selection tool. For BrdU incorporation assay, cells were treated with BrdU at a concentration of 33 μM for 2 hours, fixed with 50%methanol/50%acetone solution for 2 minutes, then processed as detailed above.
The authors declare that they have no competing interests in relation to the work described.
This work was supported by an MPP grant from the American University of Beirut, and in part by the American Lebanese Syrian Associated Charities (ALSAC) and International Outreach Program at St Jude Children’s Research Hospital, Memphis, TN. The authors thank Bristol-Myers Squibb, M. Barbacid, and M. F. Roussel for donating the p18Ink4c -/- mice; and support from the AUB Core Laboratory Shared Resources.
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