A number of small-molecule inhibitors of Aurora kinases have been developed and are undergoing clinical trials for anti-cancer therapies. Different Aurora kinases, however, behave as very different targets: while inhibition of Aurora A (AURKA) induces a delay in mitotic exit, inhibition of Aurora B (AURKB) triggers mitotic slippage. Furthermore, while it is evident that p53 is regulated by Aurora kinase-dependent phosphorylation, how p53 may in turn regulate Aurora kinases remains mysterious. To address these issues, isogenic p53-containing and -negative cells were exposed to classic inhibitors that target both AURKA and AURKB (Alisertib and ZM447439), as well as to new generation of inhibitors that target AURKA (MK-5108), AURKB (Barasertib) individually. The fate of individual cells was then tracked with time-lapse microscopy. Remarkably, loss of p53, either by gene disruption or small interfering RNA-mediated depletion, sensitized cells to inhibition of both AURKA and AURKB, promoting mitotic arrest and slippage respectively. As the p53-dependent post-mitotic checkpoint is also important for preventing genome reduplication after mitotic slippage, these studies indicate that the loss of p53 in cancer cells represents a major opportunity for anti-cancer drugs targeting the Aurora kinases.
Accurate cell division relies on a well-balanced regulating network of protein kinases and phosphatases.1 One important family of mitotic kinases is the Aurora kinases. While yeasts contain a single Aurora kinase, mammals contain three homologs that display distinctive functions, substrate specificity and cellular localization during mitosis.
Aurora A (also called AURKA) is a centrosomal protein that regulates the maturation and separation of centrosomes and formation of bipolar spindle.2 Although related in sequence to AURKA, Aurora B (also called AURKB) is a component of the chromosomal passenger complex, which comprises AURKB, INCENP, borealin and survivin. chromosomal passenger complex localizes to chromosomes and kinetochores in early mitosis and functions in chromosome–microtubule interactions, sister chromatid cohesion and the spindle-assembly checkpoint. In anaphase, the chromosomal passenger complex is relocated to the mid zone to promote cytokinesis.3 The role of the last family member, Aurora C (also called AURKC), which is mainly expressed in testis, is not well characterized.4
The activity of AURKA increases from late G2-phase onwards and peaks in prometaphase. On the other hand, the activity of AURKB peaks from metaphase to the end of mitosis. Activation of AURKA requires binding to specific cofactors including Ajuba, Bora and TPX2, leading to the autophosphorylation of a residue in the T-loop (Thr288).5 Similarly, AURKB is activated by autophosphorylation of Thr232 in the T-loop after binding to members of the chromosomal passenger complex.6 At the end of mitosis, both AURKA and AURKB are degraded by APC/C-mediated ubiquitination.
As Aurora kinases are upregulated in several human cancers and correlated with poor prognosis, they are believed to be important anti-cancer drug targets.7 A number of small-molecule Aurora kinase inhibitors have been developed over the recent years and more than 20 compounds are currently at various stages of development and clinical trials.8 While the first group of Aurora kinase inhibitors inactivates both AURKA and AURKB indiscriminately, several later generations of inhibitors are able to specifically target AURKA or AURKB.
Due to the different functions of AURKA and AURKB, the effects of their downregulation or pharmacological inactivation are different. Inhibition of AURKA causes defects in centrosome separation and spindle formation, resulting in mitotic arrest and apoptosis. By contrast, inhibition of AURKB interferes with histone H3 phosphorylation, chromosome segregation and cytokinesis, causing the formation of polyploid cells.9
Among the identified substrates for the Aurora kinases, the transcription factor p53 is one of the prominent substrates that may be involved in the checkpoint and apoptotic responses after inhibition of Aurora kinases. AURKA phosphorylates p53 at Ser215 and Ser315 and downregulates its transactivation activity and protein stability.10, 11 Hence inhibition of AURKA can promote the accumulation and activation of p53. How p53 may conversely regulate Aurora kinase is currently uncertain.
As studies on pharmacological inhibition of Aurora kinases generally focus on end-point cytotoxicity assays, how single cells behave dynamically remains incompletely understood. In this study, we have treated p53-containing and -negative cells to small inhibitors targeting AURKA and/or AURKB, and tracked the fate of individual cells with time-lapse microscopy to address these issues.
Alisertib inhibits both AURKA and AURKB and promotes mitotic exit delay and slippage
Alisertib (also called MLN8237), the first orally available Aurora kinase inhibitor to enter human clinical trials, is generally believed to be an AURKA-specific inhibitor with IC50 of 1.2 nM in vitro.12 As expected, exposure of HeLa cells to Alisertib (250 nM) induced a G2/M cell cycle delay (Figure 1a). A higher concentration (1 μM), however, resulted in a repeated round of DNA synthesis, suggesting Alisertib could also induce mitotic slippage or failure of cytokinesis.
To demonstrate more rigorous that premature mitotic exit was indeed triggered by Alisertib, the fate of individual cells were tracked with time-lapse microscopy (Figure 1b). Consistent with an inhibition of AURKA, mitosis was lengthened progressively with increasing concentration of Alisertib up to 250 nM (quantified in Figure 1c). For the cells that could exit mitosis, they underwent anaphase and cytokinesis relatively normally. By contrast, further increase in the concentrations of Alisertib resulted in a shortening of mitosis and increase in mitotic slippage (no anaphase), suggesting that AURKB could also be inhibited. Examples of control (Supplementary Video S1), Alisertib-mediated mitotic retardation (Supplementary Video S2) and mitotic slippage (Supplementary Video S3) are shown in the Supplementary Movies.
Extensive cell death was associated with the defective mitosis induced by 250 nM of Alisertib (Figure 1b). Interestingly, the shortening of mitosis due to slippage at higher concentrations of Alisertib actually increased overall cell survival within the imaging period (Figure 1b). Nevertheless, the resulting tetraploid cells die shortly afterwards, as indicated by imaging the cells for a longer period of time (Supplementary Figure S1) and the massive sub-G1 population at 48 h (Figure 1d). We also used trypan blue exclusion assays to confirm the cytotoxicity caused by the different concentrations of Alisertib (Figure 1e). Finally, the percentage of clonogenic survival after treatment with 250 nM of Alisertib was consistent with the portion of cells that eventually able to complete mitosis during live-cell imaging (∼30%; Figure 1b). By comparison, although ∼98% cells survived immediately following Alisertib (1 μM)-mediated mitotic slippage (Figure 1b), only a small portion survived in clonogenic assays (Figure 1f).
Inactivation of different Aurora kinases was detected directly using an antibody that recognized the phosphorylated forms of Aurora kinases (AURKAThr288, AURKBThr232 and AURKCThr198) (Figure 1g). As expected, AURKA and AURKB were activated during mitosis (lane 2). Alisertib inhibited AURKA and AURKB differentially: while AURKA was completely inhibited with 50 nM of Alisertib, complete inhibition of AURKB required 1 μM of Alisertib. Although the above experiment showed that Alisertib could inhibit mitotic AURKA and AURKB, cells are generally exposed to inhibitors before they enter mitosis. To better recapitulate this situation, cells were first exposed to Alisertib before nocodazole was added to trap cells in mitosis. Figure 1h confirms that while 50 nM of Alisertib was adequate to prevent AURKA activation, 1 μM of Alisertib was required to abolish AURKB activation.
In addition to AURKA and AURKB, the anti-phospho-Aurora antibody also recognized a faster-migrating band of similar size as predicted for AURKC (indicated by an asterisk in this paper). Nevertheless, several pieces of evidence suggested that the band is not AURKC (see Supplementary Figure S2). Instead, it could be an isoform or truncated version of AURKB. In all our experiments, phosphorylation of this band completely followed that of AURKB.
To consolidate the link between mitotic slippage and AURKB inhibition, we conducted similar analysis with another AURKB inhibitor called ZM447439. Similar to Alisertib, ZM447439 also inhibited both AURKA and AURKB, with IC50 of 110 nM and 130 nM, respectively.13 Although ZM447439 could also inhibit AURKA without affecting AURKB, the range of concentrations that could differentiate the two Aurora kinases was narrower compared with Alisertib (Supplementary Figure S3A). Accordingly, cells readily underwent mitotic slippage when incubated with 1 μM of ZM447439, a concentration that targeted both AURKA and AURKB (Supplementary Figure S3B).
To ascertain that p53-containing cells also responded similarly to Alisertib, we treated HCT116 cells with Alisertib and showed its ability to differentially inhibit AURKA and AURKB (Supplementary Figure S4A). Single-cell analysis confirmed the dose-dependent induction of mitotic retardation and slippage (Supplementary Figure S4B). Clonogenic survival was reduced by inhibition of AURKA alone or together with AURKB (Supplementary Figure S4C). As expected, downregulation of AURKB with small interfering RNA (siRNA) promoted Alisertib-mediated mitotic slippage, further verifying that the mitotic slippage was triggered by an inhibition of AURKB (Supplementary Figure S4B).
Collectively, these results indicate that AURKA and AURKB can be differentially inhibited by Alisertib, causing mitotic exit delay and slippage, respectively.
Specific inhibition of AURKA and AURKB induces mitotic exit delay and mitotic slippage respectively
Given that Alisertib and ZM447439 targeted both AURKA and AURKB, we next sought to resolve if mitotic slippage could be induced by inhibition of AURKB alone by using the inhibitor Barasertib (also called AZD1152-HQPA).14 Figure 2a shows that Barasertib inactivated AURKB without affecting AURKA over a range of concentrations. In agreement with this, Barasertib stimulated mitotic slippage in ∼100% of the cells (Figures 2b and c). Accordingly, whole-genome reduplication occurred after Barasertib treatment in both HeLa (Figure 2d) and HCT116 (Figure 2e). As expected, cell proliferation (as measured with WST-1 assays) was reduced after Barasertib treatment in the cell lines (Figure 2f). Upon further incubation, more cells displayed apoptosis morphology (Supplementary Figure S1) and sub-G1 DNA contents, which could be reduced with a caspase inhibitor (Figure 2g).
Interestingly, mitotic slippage induced by Barasertib was not identical to that induced by Alisertib. Alisertib (or ZM447439)-treated cells exited mitosis from a prometaphase-like state. While some Barasertib-treated cells also exited mitosis prematurely from a prometaphase-like state (Supplementary Video S4), a subset of cells were able to form a metaphase plate before DNA decondensation (Supplementary Video S5) (quantified in Supplementary Figure S5). Neither of these cell populations could undergo proper chromosome segregation and anaphase. These results indicate while metaphase plate could form when AURKB alone was inhibited, it was not formed when both AURKA and AURKB were inhibited at the same time.
Alternatively, AURKA could be specifically inactivated with the inhibitor MK-5108 (also called VX-689).15 Specific inhibition of AURKA was demonstrated by the loss of mitotic AURKAThr288 phosphorylation without affecting AURKBThr232 phosphorylation (Figure 3a). Likewise, activation of AURKA, but not AURKB, was inhibited by MK-5108 (Figure 3b). Flow cytometry analysis indicated that MK-5108 induced a G2/M delay without evidence of genome reduplication (Figure 3c), suggesting that AURKA inhibition induced a mitotic exit delay without triggering mitotic slippage. This was later confirmed more rigorously using live-cell imaging (see Figure 7c).
Collectively, these results unequivocally show that inhibition of AURKB alone (rather than together with AURKA) is sufficient to trigger mitotic slippage, resulting in extensive apoptosis.
p53 is activated by inhibitors of AURKA and AURKB
To examine the relationship between p53 and inhibition of AURKA and AURKB, several p53-containing cell lines were challenged with different Aurora inhibitors. When HCT116 cells were incubated with Alisertib, both p53 and its downstream transcriptional target p21CIP1/WAF1 were stabilized in a dose-dependent manner (Figure 4a). Notably, p53 was activated by Alisertib at concentrations that targeted AURKA or AURKB (see Supplementary Figure S4). The results were confirmed with another p53-containing cell line HepG2 (Figure 4b). The p53-dependent nature of p21CIP1/WAF1 induction was demonstrated by the fact that it was abolished in the isogenic HCT116(p53−/−) cell line (Figure 4b).
We next examined if specific inhibition of AURKA or AURKB also activates p53. Figure 4c shows both p53 and p21CIP1/WAF1 were induced after HCT116 cells were incubated with Barasertib or MK-5108. Taken together, these results indicated that p53 is stabilized and activated after treatment with Aurora inhibitors at concentrations that induced mitotic exit delay or slippage.
Loss of p53 promotes AURKB inhibition, mitotic slippage and genome reduplication
Given that p53 is activated by different Aurora inhibitors, we next examined if the mitotic responses to the Aurora inhibitors are influenced by p53. Clones of HCT116 and HCT116(p53−/−) stably expressing histone H2B-GFP were generated. The cells were examined with time-lapse microscopy after challenged with Alisertib. Figure 5a shows that both p53-containing and -deficient cells responded to high concentration of Alisertib (1 μM) similarly by undergoing mitotic slippage. However, the cells behaved very differently when treated with 250 nM of Alisertib: while HCT116 exhibited a longer mitosis typical of AURKA inhibition (Supplementary Figure S4C), a large subset of HCT116(p53−/−) underwent very protracted mitosis as well as mitotic slippage. In agreement with this, more HCT116(p53−/−) displayed 4N DNA contents after treatment with 250 nM of Alisertib than HCT116 (Figure 5b). As the post-mitotic checkpoint also relies on p53, the HCT116(p53−/−) cells also underwent genome reduplication after mitotic slippage (8N DNA contents). Also in agreement with the cytotoxicity associated with mitotic slippage (see above), HCT116(p53−/−) appeared to be more sensitive than HCT116 to Alisertib in long-term clonogenic survival (Figure 5c).
A trivial explanation of the difference in drug sensitivity of p53-containing and -deficient cells is that the p53-deficient cells contained less AURKA and AURKB than wild-type cells. To test this hypothesis, the expression of AURKA and AURKB in HCT116 and HCT116(p53−/−) was examined side-by-side by immunoblotting (Figure 5d). By quantifying the immunoblots using the Odysseus infrared imaging system, no significant difference in AURKA was found between the two cell lines and a twofold increase in AURKB was found in the p53-deficient cells (data not shown). Furthermore, phosphorylation of AURKAThr288 and AURKBThr232 was inhibited similarly in the presence or absence of p53 (Figure 5e), suggesting that the differential response was unlikely to be due to the Aurora kinases per se.
Although HCT116 and HCT116(p53−/−) are isogenic cell lines, the fact that p53 is essential for maintaining genome stability opens the possibility that many genetic alterations may have occurred during culturing. To avoid the indirect effects due to the long-term p53 deficiency, we also performed experiments in which p53 was transiently depleted with siRNAs (Figure 6a). Figure 6b shows that p53-depleted HCT116 cells underwent mitotic slippage at lower concentrations of Alisertib than control-depleted cells. Effective depletion of p53 was confirmed by immunoblotting (Figure 6c). Of note is that the expression of AURKA and AURKB was not affected by the p53 siRNA. These results indicated that the sensitivity to Alisertib-mediated mitotic slippage was also increased in cells transiently depleted of p53.
As Alisertib inhibited both AURKA and AURKB, we next investigated whether p53 deficiency sensitized cells specifically to AURKB inhibition. HCT116 cells were transfected with control or p53 siRNAs before incubating with different concentrations of Barasertib. Figure 7a shows that while only ∼20% of control cells underwent mitotic slippage in the presence of 6.25 nM of Barasertib, ∼60% of p53-depleted cells underwent mitotic slippage with the same treatment (summarized in Figure 7b).
We also depleted p53 in another cell line (HepG2) to verify these results. Once again the abundance of AURKA or AURKB was not affected by the absence of p53 (Figure 6c). Single-cell analysis revealed that p53-depleted HepG2 cells were more prone to mitotic slippage after Barasertib treatment than control cells (Supplementary Figure S6).
Collectively, these data indicate that cells lacking p53, either by gene disruption or siRNA-mediated depletion, are sensitized to AURKB inhibitors, resulting in mitotic slippage and subsequent genome reduplication.
Loss of p53 promotes AURKA inhibition and mitotic defects
In addition to inducing mitotic slippage, Alisertib also delayed mitotic exit in p53-deficient cells (Figure 4a), suggesting that AURKA inactivation was also sensitized by p53 deficiency. To test this directly, we treated p53-proficient and -deficient cells with the AURKA-specific inhibitor MK-5108. As expected, incubation with MK-5108 lengthened the mitosis of HCT116 cells in a dose-dependent manner (Figure 7c). Consistent with the specific inhibition of AURKA, mitotic slippage was not induced. Significantly, lower concentrations of MK-5108 were sufficient to delay mitotic exit in HCT116(p53−/−) cells than in control HCT116 cells (quantified in Figure 7d). Collectively, these results indicate that the loss of p53 sensitizes cells to the inhibition of both AURKA and AURKB.
Given that inhibition of AURKA and AURKB causes essentially opposite effects (mitotic exit delay and mitotic slippage, respectively), the two Aurora kinases in fact represent two different anti-cancer drug targets. We used an antibody that recognized phosphorylated AURKAThr288 and AURKBThr232 to determine the activation (Figure 1h) or inactivation (Figure 1g) of the Aurora kinases. These analyses could differentiate the actions of different Aurora kinase inhibitors. Pan-Aurora kinase inhibitor that inhibited both AURKA and AURKB was exemplified by ZM447439 (Supplementary Figure S3). Although Alisertib was also a pan-Aurora kinase inhibitor, it inhibited either AURKA alone or AURKA and AURKB simultaneously in a concentration-dependent manner (Figures 1g and h). By contrast, newer generation of inhibitors such as MK-5108 (Figure 3) or Barasertib (Figure 2a) could achieve specific inhibition of AURKA or AURKB, respectively.
One conclusion from experiments using these small-molecule inhibitors is that inhibition of AURKB alone (rather than together with AURKA) is sufficient to trigger mitotic slippage, indicating that mitotic slippage does not require AURKA to be first inhibited. This is in good agreement with studies using other pan-Aurora inhibitors including VX-68016 and ZM447439.17 However, the mechanism leading to mitotic slippage appears to be different when AURKA was also inhibited, because some cells could form a metaphase plate after AURKB alone was inhibited with Barasertib (Supplementary Video S5). Interestingly, the precise effects of inhibition of AURKB by Barasertib may be cell line-specific. In contrast to HeLa (Figure 2) and HCT116 cells (data not shown), which underwent mitotic slippage in the presence of Barasertib, HONE1 cells (nasopharyngeal carcinoma) underwent chromosome segregation before failing cytokinesis, giving rise to binucleated tetraploid cells (Jinny Hong and RYCP, unpublished data). This is in agreement from studies using chicken DT40 cells, in which cells lacking AURKB can form bipolar spindles but fail to properly align their chromosomes and exit mitosis with cytokinesis defects. Only when both AURKA and AURKB were deleted, cells exit mitosis without anaphase.18
Whether AURKA or AURKB is the better anti-cancer drug target is debatable.9 This may not be such a critical consideration in the past due to the fact that early generations of Aurora kinase inhibitors are generally not very specific and target both AURKA and AURKB. And as discussed above, this essentially promotes mitotic slippage followed by apoptosis. With the more recent development of relative specific inhibitors, this question has become relevant, in particular for treatment of cancer cells that have different propensity in undergoing mitotic slippage.
Compared with inhibition of AURKB, inhibition of AURKA perhaps has an advantage of eliminating cancer cells quickly during the mitotic block. Nevertheless, as can be seen after HeLa cells were treated with 250 nM of Alisertib (Figures 1b and Supplementary Figure S1), only a portion of cells underwent apoptosis during mitosis. The rest (10–30%) were eventually able to complete mitosis. This proportion was consistent with the percentage of clonogenic survival after Alisertib treatment (Figure 1f).
Mitotic cell death was even less pronounced in HCT116 than in HeLa after treatment with concentrations of Alisertib that inhibited AURKA (Supplementary Figure S4C) or with the AURKA-specific MK-5108 (Figure 7c). Nevertheless, HCT116 cells were very sensitive to AURKA inhibition as measured with clonogenic survival (Supplementary Figure S4B). The discrepancy was likely to be due to the activation of p53 by the AURKA inhibitors (Figure 4), which possibly could trigger cell cycle arrest and apoptosis after the initial mitotic arrest. In support of this, the p53 downstream target p21CIP1/WAF1 was induced after treatment with Alisertib or MK-5108 (Figure 4).
Inhibition of AURKB, in contrast, triggers mitotic slippage and tetraploidization. A p53-dependent ‘tetraploidy checkpoint’ has been proposed to prevent S-phase entry in cells that have undergone mitotic slippage or aborted cytokinesis.19 The checkpoint is implicated to sense the increase in chromosome number and halt the cell in a tetraploid G1 state. However, the existence of this checkpoint has been disputed.20, 21, 22 It is likely that the p53-dependent arrest after tetraploidization is mainly due to DNA damage or centrosomal stress during the aberrant mitosis.23
Irrespective of the actual signals that activate the checkpoint, p53 was clearly activated by Aurora kinase inhibitors that triggered mitotic slippage (Figure 4). This may at least explain why despite the fact that ∼90% of HCT116 cells survived after mitotic slippage (for example, Supplementary Figure S4C), clonogenic survival was only at ∼20% (Supplementary Figure S4B). How inhibition of Aurora kinases activates p53 is not completely understood. As AURKA has been shown to directly phosphorylate p53 and downregulate its transactivation activity and protein stability, AURKA inhibitors are expected to promote the accumulation and activity of p53.10, 11 AURKB has not been shown to regulate p53 directly. Instead of direct action, it is possible that stress during the aberrant mitosis caused by AURKB inhibition may be involved in activating p53.
Nevertheless, p53-dependent mechanism was clearly not the only mechanism that suppressed cell growth after mitotic slippage. For example, HeLa cells also have low clonogenic survival after Alisertib-induced mitotic slippage (Figure 1f). This p53-independent cytotoxicity was likely to be contributed by the genome reduplication and subsequent multipolar mitosis after mitotic slippage (Figures 1a–d).
Collectively, our data suggest that the status of p53 affects the effectiveness of AURKA- and AURKB-based therapies differently. As both AURKA- and AURKB-specific inhibitors activate p53, cell cycle arrest and apoptosis are major treatment outcomes for p53-positive cells. For p53-negative cells treated with AURKB inhibitors, however, genome reduplication and multipolar mitosis after mitotic slippage become unchecked, and many cells are then eliminated due to gross chromosomal instability. Following these arguments, AURKB inhibitors should be effective for both p53-positive and -negative cells. On the other hand, the lack of p53 promotes the survival of cells that can complete mitosis after treatment with AURKA inhibitors. But it is also possible that other mechanisms, such as p73, are involved in apoptosis after AURKA inhibition in p53-deficient cells.24
In this study, we found that in addition to its role in cell cycle arrest and apoptosis, p53 also affected the intrinsic sensitivity to Aurora kinase inhibitors. Compare to HCT116 cells, HCT116(p53−/−) cells were more sensitive to the AURKA inhibitor MK-5108 (Figure 7c) and the AURKB inhibitor Barasertib (Figure 7a). The main effects of inhibition of the individual Aurora kinase, namely mitotic exit delay or mitotic slippage, were induced by a lower drug concentration in HCT116(p53−/−) cells. Consistent with these results, HCT116(p53−/−) cells were more sensitive than HCT116 to Alisertib-induced mitotic exit delay and slippage (Figure 5a) and in long-term clonogenic survival (Figure 5c). Similar results were obtained when p53 was depleted with siRNAs (Figure 6a and Supplementary Figure S6), excluding the possibility that the p53-dependent effects were resulted from long-term genome instability. Similar results were also obtained when we imaged the cells using bright field only (using re-attachment as an indicator of mitotic slippage), excluding the possibility that UV stress of the imaging may contribute to the p53-dependent responses (our unpublished data).
Why a loss of p53 increases in sensitivity to Aurora kinase inhibitors? The expression of AURKA and AURKB was not affected after p53 was disrupted with homologous recombination or depletion with siRNAs (Figure 6c). Inactivation of individual Aurora kinases also appeared to be unaffected by p53 (Figure 5e). In fact, the answer to the question may not be straightforward due to the myriad of transcriptional targets of p53 and the equally numerous substrates of Aurora kinases. Several p53 targets including GADD45a have been reported to interact with Aurora kinases.25 However, we did not detect differences in GADD45a expression in p53-containing and -deficient cells with or without Aurora kinase inhibition (our unpublished data). Likewise, knockdown of the p53 target p21CIP1/WAF1 did not affect responses to Aurora kinase inhibitors (our unpublished data).
Although the molecular basis of p53-dependent sensitivity to Aurora kinase inhibitors is not known, the fact that p53 is mutated in half of all cancers has important implications in therapies targeting Aurora kinases. Several published studies have also hinted the involvement of p53 in the sensitivity to Aurora inhibitors. For example, Barasertib-mediated radiosensitization is more pronounced in p53-negative cancer cells than in p53-positive cells.26 The induction of endoreduplication and apoptosis by VX-680 was also more effective in cells lacking the p53 post-mitotic checkpoint.27 Other studies, however, did not observe an effect of p53 on Aurora kinase inhibitors.28 It should be noted that the published studies are mainly based on end-point assays. Only the extensive use of live-cell imaging allowed us to identify the increase in mitotic exit delay and slippage in the absence of p53, which may also explain why these discoveries have not been made earlier.
In conclusion, loss of p53 increases sensitivity to pharmacological inhibition of both AURKA and AURKB, causing mitotic exit delay and slippage, respectively. As the p53 post-mitotic checkpoint is also important for preventing genome reduplication after mitotic slippage, p53 deficiency represents a ‘double whammy’ for cancer cells treated with Aurora kinase inhibitors.
Materials and methods
All reagents were obtained from Sigma-Aldrich (St Louis, MO, USA) unless stated otherwise.
HepG2 was obtained from the American Type Culture Collection (Manassas, VA, USA). HCT116 (colorectal carcinoma) and HCT116(p53−/−) were gifts from Dr Bert Vogelstein (Johns Hopkins University). No authentication was done by the authors. The HeLa used in this study was a clone that expressed the tTA tetracycline repressor chimera.29 HeLa30 and HCT11631 that stably expressed histone H2B-green fluorescent protein were used for live-cell imaging. Similar approach was used to generate HCT116(p53−/−) that expressed histone H2B-green fluorescent protein. Cells were propagated in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) calf serum (Life Technologies, Carlsbad, CA, USA) (for HeLa) or fetal bovine serum (Life Technologies) (for HCT116 and HepG2) and 50 U/ml penicillin streptomycin (Life Technologies) in a humidified incubator at 37 °C in 5% CO2. Unless stated otherwise, cells were treated with the following reagents at the indicated final concentration: Alisertib (Selleck Chemicals, Houston, TX, USA) (1 μM), Barasertib (Selleck Chemicals) (25 nM), MG132 (10 μM), nocodazole (0.1 μg/ml for HeLa; 0.4 μg/ml for HCT116), thymidine (2 mM), MK-5108 (Selleck Chemicals) (1 μM), ZM447439 (Selleck Chemicals) (2 μM) and Z-VAD(OMe)-FMK (Enzo Life Sciences, Farmingdale, NY, USA) (10 μM). Synchronization at mitosis was performed by first releasing cells from a double thymidine block32 for 6 h before adding nocodazole for another 6 h; mitotic cells were then collected by mechanical shake off. Cell-free extracts were prepared as described previously.33
siRNA and transfection
Cells were transfected with siRNA by Lipofectamine RNAiMAX (Life Technologies). The following siRNAs were obtained from the indicated suppliers: AURKA 5′-IndexTermGGCCAAUGCUCAGAGAAGUACUUGA-3′), AURKB (5′-IndexTermUCUUAGGGCUCAAGGGAGAGCUGAA-3′), p53 (a mixture of three siRNA was used: 5′-IndexTermGCUUCGAGAUGUUCCGAGAGCUGAA-3′, 5′-IndexTermCCGGACGAUAUUGAACAAUGGUUCA-3′ and 5′-IndexTermGCCAAGUCUGUGACUUGCACGUACU-3′) (Life Technologies); AURKC (5′-IndexTermGAUCCAGGCUCAUCUACAA-3′) (RiboBio, Guangzhou, China).
Cell viability assays
Trypan blue analysis was performed as described.34 WST-1 assays were performed according to the instructions of the manufacturer (Roche Applied Science, Indianapolis, IN, USA). For clonogenic survival assays, 1000 cells were seeded onto 60 mm-dishes. After 12 h, the cells were either mock treated or exposed to the different concentrations of drugs for another 24 h. The cells were then gently washed three times with phosphate-buffered saline and cultured in normal medium. After 10 days, colonies were fixed with methanol/acetic acid (2:1 v/v) and visualized by staining with 2% (w/v) crystal violet in 20% methanol.
Flow cytometry analysis after propidium iodide staining was performed as described previously.34
Cells were seeded onto poly-lysine-coated glass plates and imaged using a TE2000E-PFS inverted fluorescent microscope, Plan Fluor ELWD ADL 20X objective N.A. 0.45 (Nikon, Melville, NY, USA) equipped with a 1 K × 1 K, 8 μm2 pixels, cooling SPOT BOOST EMCCD camera (Diagnostic Instrument, Sterling Heights, MI, USA) and a INU-NI- F1 temperature, humidity, and CO2 control system (Tokai Hit, Shizuoka-ken, Japan). enhanced green fluorescent protein was excited by 12.5% output of HG Precentered Fiber Illuminator (Nikon) with HQ470/30 excitation filter (Chroma, Bellows Falls, VT, USA). The fluorescent images were acquired with minimal exposure time (50–200 ms). Data acquisition was carried out at 5 m/frame.
Antibodies and immunological methods
Antibodies against β-actin,35 CDK136 and cyclin B130 were obtained from sources as described previously. Antibodies against phospho-histone H3Ser10, p21CIP1/WAF1, p53 and PLK1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), AURKA (BD Biosciences, Franklin Lakes, NJ, USA), AURKB (Sigma-Aldrich), AURKC (Life Technologies) and phospho-AURKAThr288/AURKBThr232/AURKCThr198 (Cell Signaling Technology, Beverly, MA, USA) were obtained from the indicated suppliers. Immunoblotting was performed as described.33
Ma HT, Poon RY . How protein kinases co-ordinate mitosis in animal cells. Biochem J 2011; 435: 17–31.
Karthigeyan D, Prasad SB, Shandilya J, Agrawal S, Kundu TK . Biology of Aurora A kinase: implications in cancer manifestation and therapy. Med Res Rev 2010; 31: 757–793.
Ruchaud S, Carmena M, Earnshaw WC . Chromosomal passengers: conducting cell division. Nat Rev Mol Cell Biol 2007; 8: 798–812.
Kimura M, Matsuda Y, Yoshioka T, Okano Y . Cell cycle-dependent expression and centrosome localization of a third human aurora/Ipl1-related protein kinase, AIK3. J Biol Chem 1999; 274: 7334–7340.
Scutt PJ, Chu ML, Sloane DA, Cherry M, Bignell CR, Williams DH et al. Discovery and exploitation of inhibitor-resistant aurora and polo kinase mutants for the analysis of mitotic networks. J Biol Chem 2009; 284: 15880–15893.
Yasui Y, Urano T, Kawajiri A, Nagata K, Tatsuka M, Saya H et al. Autophosphorylation of a newly identified site of Aurora-B is indispensable for cytokinesis. J Biol Chem 2004; 279: 12997–13003.
Gautschi O, Heighway J, Mack PC, Purnell PR, Lara PNJ, Gandara DR . Aurora kinases as anticancer drug targets. Clin Cancer Res 2008; 14: 1639–1648.
Green MR, Woolery JE, Mahadevan D . Update on Aurora kinase targeted therapeutics in oncology. Expert Opin Drug Discov 2011; 6: 291–307.
Keen N, Taylor S . Aurora-kinase inhibitors as anticancer agents. Nat Rev Cancer 2004; 4: 927–936.
Liu Q, Kaneko S, Yang L, Feldman RI, Nicosia SV, Chen J et al. Aurora-A abrogation of p53 DNA binding and transactivation activity by phosphorylation of serine 215. J Biol Chem 2004; 279: 52175–52182.
Katayama H, Sasai K, Kawai H, Yuan ZM, Bondaruk J, Suzuki F et al. Phosphorylation by aurora kinase A induces Mdm2-mediated destabilization and inhibition of p53. Nat Genet 2004; 36: 55–62.
Manfredi MG, Ecsedy JA, Chakravarty A, Silverman L, Zhang M, Hoar KM et al. Characterization of Alisertib (MLN8237), an investigational small-molecule inhibitor of aurora A kinase using novel in vivo pharmacodynamic assays. Clin Cancer Res 2011; 17: 7614–7624.
Ditchfield C, Johnson VL, Tighe A, Ellston R, Haworth C, Johnson T et al. Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores. J Cell Biol 2003; 161: 267–280.
Yang J, Ikezoe T, Nishioka C, Tasaka T, Taniguchi A, Kuwayama Y et al. AZD1152, a novel and selective aurora B kinase inhibitor, induces growth arrest, apoptosis, and sensitization for tubulin depolymerizing agent or topoisomerase II inhibitor in human acute leukemia cells in vitro and in vivo. Blood 2007; 110: 2034–2040.
Shimomura T, Hasako S, Nakatsuru Y, Mita T, Ichikawa K, Kodera T et al. MK-5108, a highly selective Aurora-A kinase inhibitor, shows antitumor activity alone and in combination with docetaxel. Mol Cancer Ther 2010; 9: 157–166.
Harrington EA, Bebbington D, Moore J, Rasmussen RK, Ajose-Adeogun AO, Nakayama T et al. VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo. Nat Med 2004; 10: 262–267.
Girdler F, Gascoigne KE, Eyers PA, Hartmuth S, Crafter C, Foote KM et al. Validating Aurora B as an anti-cancer drug target. J Cell Sci 2006; 119: 3664–3675.
Hegarat N, Smith E, Nayak G, Takeda S, Eyers PA, Hochegger H . Aurora A and Aurora B jointly coordinate chromosome segregation and anaphase microtubule dynamics. J Cell Biol 2011; 195: 1103–1113.
Andreassen PR, Lohez OD, Lacroix FB, Margolis RL . Tetraploid state induces p53-dependent arrest of nontransformed mammalian cells in G1. Mol Biol Cell 2001; 12: 1315–1328.
Fujiwara T, Bandi M, Nitta M, Ivanova EV, Bronson RT, Pellman D . Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells. Nature 2005; 437: 1043–1047.
Uetake Y, Sluder G . Cell cycle progression after cleavage failure: mammalian somatic cells do not possess a "tetraploidy checkpoint". J Cell Biol 2004; 165: 609–615.
Wong C, Stearns T . Mammalian cells lack checkpoints for tetraploidy, aberrant centrosome number, and cytokinesis failure. BMC Cell Biol 2005; 6: 6.
Storchova Z, Kuffer C . The consequences of tetraploidy and aneuploidy. J Cell Sci 2008; 121: 3859–3866.
Dar AA, Belkhiri A, Ecsedy J, Zaika A, El-Rifai W . Aurora kinase A inhibition leads to p73-dependent apoptosis in p53-deficient cancer cells. Cancer Res 2008; 68: 8998–9004.
Shao S, Wang Y, Jin S, Song Y, Wang X, Fan W et al. Gadd45a interacts with aurora-A and inhibits its kinase activity. J Biol Chem 2006; 281: 28943–28950.
Tao Y, Zhang P, Girdler F, Frascogna V, Castedo M, Bourhis J et al. Enhancement of radiation response in p53-deficient cancer cells by the Aurora-B kinase inhibitor AZD1152. Oncogene 2008; 27: 3244–3255.
Gizatullin F, Yao Y, Kung V, Harding MW, Loda M, Shapiro GI . The Aurora kinase inhibitor VX-680 induces endoreduplication and apoptosis preferentially in cells with compromised p53-dependent postmitotic checkpoint function. Cancer Res 2006; 66: 7668–7677.
Curry J, Angove H, Fazal L, Lyons J, Reule M, Thompson N et al. Aurora B kinase inhibition in mitosis: strategies for optimising the use of aurora kinase inhibitors such as AT9283. Cell Cycle 2009; 8: 1921–1929.
Yam CH, Siu WY, Lau A, Poon RY . Degradation of cyclin A does not require its phosphorylation by CDC2 and cyclin-dependent kinase 2. J Biol Chem 2000; 275: 3158–3167.
Chan YW, Ma HT, Wong W, Ho CC, On KF, Poon RY . CDK1 inhibitors antagonize the immediate apoptosis triggered by spindle disruption but promote apoptosis following the subsequent rereplication and abnormal mitosis. Cell Cycle 2008; 7: 1449–1461.
On KF, Chen Y, Ma HT, Chow JP, Poon RY . Determinants of mitotic catastrophe on abrogation of the G2 DNA damage checkpoint by UCN-01. Mol Cancer Ther 2011; 10: 784–794.
Ma HT, Poon RY . Synchronization of HeLa cells. Methods Mol Biol 2011; 761: 151–161.
Poon RY, Toyoshima H, Hunter T . Redistribution of the CDK inhibitor p27 between different cyclin.CDK complexes in the mouse fibroblast cell cycle and in cells arrested with lovastatin or ultraviolet irradiation. Mol Biol Cell 1995; 6: 1197–1213.
Siu WY, Arooz T, Poon RY . Differential responses of proliferating versus quiescent cells to adriamycin. Exp Cell Res 1999; 250: 131–141.
Chan YW, On KF, Chan WM, Wong W, Siu HO, Hau PM et al. The kinetics of p53 activation versus cyclin E accumulation underlies the relationship between the spindle-assembly checkpoint and the postmitotic checkpoint. J Biol Chem 2008; 283: 15716–15723.
Siu WY, Lau A, Arooz T, Chow JP, Ho HT, Poon RY . Topoisomerase poisons differentially activate DNA damage checkpoints through ataxia-telangiectasia mutated-dependent and -independent mechanisms. Mol Cancer Ther 2004; 3: 621–632.
Many thanks are due to Michelle Chen, Nelson Lee and Kenji Nishiura for technical assistance. This work was supported in part by the Research Grants Council grant HKU7/CRG/09 to RYCP.
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on the Oncogene website
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Marxer, M., Ma, H., Man, W. et al. p53 deficiency enhances mitotic arrest and slippage induced by pharmacological inhibition of Aurora kinases. Oncogene 33, 3550–3560 (2014) doi:10.1038/onc.2013.325
- mitotic catastrophe
- mitotic slippage
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