Antagonizing the spindle assembly checkpoint silencing enhances paclitaxel and Navitoclax-mediated apoptosis with distinct mechanistic

Antimitotic drugs arrest cells in mitosis through chronic activation of the spindle assembly checkpoint (SAC), leading to cell death. However, drug-treated cancer cells can escape death by undergoing mitotic slippage, due to premature mitotic exit. Therefore, overcoming slippage issue is a promising chemotherapeutic strategy to improve the effectiveness of antimitotics. Here, we antagonized SAC silencing by knocking down the MAD2-binding protein p31comet, to delay mitotic slippage, and tracked cancer cells treated with the antimitotic drug paclitaxel, over 3 days live-cell time-lapse analysis. We found that in the absence of p31comet, the duration of mitotic block was increased in cells challenged with nanomolar concentrations of paclitaxel, leading to an additive effects in terms of cell death which was predominantly anticipated during the first mitosis. As accumulation of an apoptotic signal was suggested to prevent mitotic slippage, when we challenged p31comet-depleted mitotic-arrested cells with the apoptosis potentiator Navitoclax (previously called ABT-263), cell fate was shifted to accelerated post-mitotic death. We conclude that inhibition of SAC silencing is critical for enhancing the lethality of antimitotic drugs as well as that of therapeutic apoptosis-inducing small molecules, with distinct mechanisms. The study highlights the potential of p31comet as a target for antimitotic therapies.

The fate of mitotic-arrested cells was reported to be dictated by two competing networks 13 . One network determines cell death through accumulation of apoptotic signals during mitosis. The other network determines mitotic slippage through gradual degradation of cyclin B1. The network that reaches its threshold first determines the cell fate. Therefore, theoretically, it should be possible to have a control over the cell fates and influence the effectiveness of antimitotics if mitotic slippage is retarded and/or death signal accumulation is accelerated 3 .
In this context, and given its key role in SAC silencing and mitotic exit, p31 comet appears as an ideal target to delay mitotic slippage. On the other hand, the BH3-only proteins, Bim, Bid, Bad and Noxa, have been shown to contribute to death in mitosis [14][15][16][17] . Thus, using BH3-mimetic drugs, in a background of mitotic slippage delay, should shift the fate of mitosis-arrested cells in favor of death. Therefore, we tested these two possibilities by monitoring cell fates by single-cell tracking during three day live-cell time-lapse analysis. Firstly, we investigated the relative contribution of delaying mitotic slippage (through p31 comet depletion) to cell death following exposure to nanomolar concentrations of paclitaxel. Secondly, we determined the relative contribution of BH3-mimeticmediated apoptosis potentiation to cell death of cells delayed in mitosis by p31 comet depletion. Results p31 comet expression and knockdown. In order to obtain a better understanding on the relevance of p31 comet as a potential target for cancer therapy, p31 comet expression was assessed in three non-small lung cancer cell lines (NSCLC): NCI-H460, A549 and Calu-3, and compared to the non-tumor cell line HPAEpiC. Upregulation of p31 comet was observed both at mRNA and protein levels in all the NSCLC cells tested comparatively to HPAEpiC (Fig. 1a,b). This highlights the importance of targeting p31 comet . Due to its suitability for quantitative evaluation of morphological changes in in vivo microscopy assays, NCI-H460 cell line was selected in this study. p31 comet knockdown was performed using siRNA duplexes previously validated 18 and ascertained by qRT-PCR and immunoblotting against p31 comet . More than 70% depletion of p31 comet was achieved, both at mRNA and protein levels, 24 h after treatment of NCI-H460 cells with p31 comet siRNAs (sip31 comet ), comparatively to cells treated with a negative control siRNA (Control siRNA) (Fig. 1c,d). These depletion levels were not altered by extended transfection time. Furthermore, contrast-phase microscopy analysis revealed an accumulation of round shaped mitotic cells and an increase in the mitotic index (Fig. 1e), in accordance with previously reported p31 comet depletion phenotype 19,20 . p31 comet depletion enhances lethality of nanomolar concentrations of paclitaxel by promoting massive cell death in mitosis. We explored whether delaying mitotic slippage, by antagonizing SAC silencing through p31 comet depletion, could potentiate cancer cell killing to nanomolar concentrations of paclitaxel, ranged from 0 to 100 nM. This is relevant as paclitaxel is used as first line chemotherapy for various cancers 1,21 . We found that paclitaxel concentrations ≥ 50 nM were needed to induce a significant increase in cytotoxicity (p < 0.0001), as determined by a 48 h MTT assays (Fig. 2a). In contrast, in cells depleted of p31 comet , concentrations as low as 10 nM of paclitaxel were sufficient to significantly reduce cell viability (p < 0.01). Indeed, dose response curves showed a threefold decrease in IC50 values for paclitaxel in cells depleted of p31 comet (Fig. 2b). Importantly, in a 10 days clonogenic assay, p31 comet knockdown significantly affected NCI-H460 cell proliferation at much lower concentrations (4 nM) of paclitaxel (p < 0.0001), suggesting that long-term survival becomes compromised at very low concentrations of paclitaxel (Fig. 2c). These results indicate that antagonizing SAC silencing by targeting p31 comet can enhance lethality of cancer cells in the presence of low doses of paclitaxel. According to the data outlined in (Fig. 2a,b), we selected the concentration of 10 nM paclitaxel to further analyze the mechanism of its combination with p31 comet suppression. It represents the lowest concentration that still leads to maximal antitumor effect when combined with p31 comet suppression. From a therapeutic point of view, this is expected to reduce paclitaxel toxicity and resistance concerns.
In order to get insight into the mechanism underlying the lethality enhancement resulting from combining p31 comet -depletion with clinically relevant concentrations of paclitaxel, we first determined the mitotic index by phase-contrast microscopy and flow cytometry. We observed an increase in the mitotic index in p31 comet -depleted cells treated with paclitaxel for 24 h (23.92 ± 2.16%), when compared to untreated (1.33 ± 0.31%) and to paclitaxeltreated cells (6.00 ± 5.1%) (Fig. 3a). Flow cytometry analysis not only confirmed these results ( Fig. 3b) but also revealed an increased sub-G1 population in p31 comet -depleted cells treated with 10 nM paclitaxel for 48 h, indicative of massive cell death. This suggests that p31 comet depletion is acting as a coadjuvant of paclitaxel, namely at 10 nM, by retaining cells in mitosis through preventing SAC silencing and, thus, delaying mitotic slippage, which may explain the enhancement of cell killing obtained in the above cytotoxic assays.
Then, we scored the duration of mitosis and the survival fate of each mitotic NCI-H460 cell by single-cell time-lapse imaging. For survival fate analysis, we considered three categories: cell death in mitosis (DiM), postmitotic death (PMD), and survivors 13 . Control and p31 comet -depleted cells were incubated with a sublethal dose (10 nM) of paclitaxel and imaged over a 72 h time course. We found that mitosis lasted 155.00 ± 199.86 min in paclitaxel-treated (n = 30), and 150.40 ± 295.99 min in p31 comet -depleted (n = 30) cells, more than four times longer than control siRNA-cells (31.33 ± 4.34 min, n = 30) (Fig. 3c). Notably, combination of p31 comet -depletion + paclitaxel resulted in a dramatic increase in mitosis duration (443.67 ± 365.92 min, n = 30), compared to control and to individual treatments (Fig. 3c), again suggesting that p31 comet downregulation, by preventing SAC silencing, delays mitotic slippage and retains low dose paclitaxel-treated cells in mitosis.
Cell survival profiling showed that the lethality of paclitaxel was increased after p31 comet -depletion ( Fig. 4a-c, and Supplementary Videos S1, S2, S3 and S4). 10 nM paclitaxel resulted in only 56.67% cell death. At this low concentration, while only a small fraction underwent DiM (16.67%) or PMD (40.00%) after one to three cycles, the remainder (43.33%) continued cycling. After p31 comet -depletion, while 13 www.nature.com/scientificreports/   www.nature.com/scientificreports/ first or second mitosis, while the survivors (23.08%) remained at interphase suggesting cell cycle arrest. Notably, combining paclitaxel with p31 comet depletion shifted the fate to DiM during the first (80%) mitosis and, interestingly, time to death was accelerated by 5.94 h and by 0,92 h comparatively to that of DiM in p31 comet -depleted cells and paclitaxel-treated cells, respectively (Fig. 4d). In the few PMD events that occurred, time to death was significantly shortened, comparatively to individual treatments. Apoptosis was the main mechanism of cell death as confirmed by TUNEL assay and Annexin-V/propidium iodide costaining (Fig. 4e,f). Overall, the results indicate that suppression of p31 comet prevents SAC silencing and delays mitotic slippage, thereby enhancing and accelerating cell death during the first mitosis, at clinically relevant doses of paclitaxel. Because the effect of the combination is close to the sum of the single effects, we conclude that the combined treatment has an additive effect. p31 comet -siRNA mediated cell death can be accelerated by a BH3-mimetic drug. Variations in cell death sensitivity to antimitotics was attributed to two competitive and mutually exclusive networks, one controlling mitotic cell death through accumulation of apoptotic signals, and the other controlling mitotic slippage through gradual cyclin B1 degradation 22 . Thus, one way to force mitosis-arrested cells to die, rather than to slip, is to challenge them with small molecules that artificially stimulate apoptosis. We thought that by delaying premature SAC silencing and, simultaneously, stimulating apoptosis signal accumulation, one should create maximal conditions for maximal cytotoxicity. We explored this possibility by combining p31 comet knockdown with the BH3-mimetic drug Navitoclax, an antagonist of the Bcl-2 family of antiapoptotic proteins Bcl-2, Bcl-XL, and Bcl-w 23 . Mitotic duration and cell fate were analyzed by live-time imaging, over 72 h experiments, as above.
First, we observed that addition of Navitoclax further compromised long-term survival of cells depleted of p31 comet (Fig. 5a). As shown in Fig. 5b, exposure to 3.5 µM Navitoclax alone did not alter mitosis duration in control siRNA cells. Interestingly, addition of Navitoclax to p31 comet siRNA transfected cells significantly reduced the duration of the mitotic block to 61.00 ± 65.51 min (n = 30), more than two times shorter compared to p31 comet siRNA transfected only cells (150.40 ± 295.99 min (n = 30). Because no mitotic role was described, so far, for the antiapototic proteins targeted by Navitoclax, we believe that the shortening of the observed mitotic arrest time is the result of precocious cell death, rather than a genuine reduction in mitotic arrest duration.
As to cell survival profile (Fig. 5c,d), Navitoclax alone induced PMD in 96.67% of treated cells, most of which (86.21%) occurred only after the second cell cycle, with an average of 7.81 h between mitotic exit and death (Fig. 5e). When Navitoclax was added to p31 comet -depleted cells, PMD after the first mitosis became the predominate cell fate (56.67%) (Fig. 5d), with death onset time significantly accelerated by approximately 8 h and 4 h, compared to Navitoclax (p < 0.05) and p31 comet -depletion individual treatments, respectively ( Fig. 5f,g, and Supplementary Videos S5 and S6). Interestingly, although some PMD (41.38%) still occurred only after the second cell cycle (Fig. 5c,d), the death onset time was significantly accelerated by approximately 1.19 h relatively to Navitoclax alone, and 5.06 h relatively to p31 comet -depletion alone (Fig. 5f), indicating that cells that escape cell death after the first cell cycle are committed to death after the second cell cycle.
In sum, the data demonstrate that the use of a BH3-mimetic in an antagonized SAC silencing background enhances and accelerates cancer cell death, largely by post-mitotic cell death after the first division.

Discussion
In this study we demonstrated that modulating SAC silencing, by p31 comet depletion, can influence mitotic slippage and cell death in the presence of spindle poisons or apoptosis potentiators. While both spindle poisons and apoptosis potentiators exacerbate cell death in p31 comet -depleted cells, they behave differently with regard to their mechanism. Depletion of p31 comet extends the duration of mitotic block in the presence of paclitaxel, and shifts cell fate to accelerated cell death in the first mitosis. In contrast, p31 comet depletion shifts cell fate to accelerated post-mitotic death after the first cell cycle, in the presence of the apoptotic potentiator Navitoclax. Thus, in both contexts, cell death is enhanced and accelerated comparatively to individual treatment. We also show that p31 comet is overexpressed in NSCLC cell lines. Our data highlight the relevance of p31 comet as drug target for cancer therapy.
We demonstrated that that p31 comet targeting enhances cancer cells exposed clinically relevant doses of paclitaxel, in an additive manner. Upon p31 comet depletion plus paclitaxel, cells were trapped in mitosis and arrested until death. This is in concordance with a previous work showing that p31 comet depletion promoted an increased duration of mitosis in the presence of paclitaxel that culminated with cell death in a colorectal carcinoma cell line 2 . However, this result was achieved under a higher concentration of paclitaxel than that used in our study. Paclitaxel inhibits tubulin depolymerization, impairing microtubule dynamics and leading to the permanent activation of SAC 3 . However, SAC can be satisfied under low concentrations of paclitaxel, allowing mitosis to proceed in the presence of spindle abnormalities and congregation errors, which may increase the aggressiveness of malignant cells 24,25 . Thus, by delaying SAC silencing, p31 comet knockdown enhances and accelerates cell death in the presence of low concentrations of paclitaxel, most probably by delaying mitotic slippage which favors accumulation of apoptotic signals. Noteworthy, the precise mechanism of paclitaxel cytotoxicity is still debatable. In addition to its antimitotic-mediated chemotherapeutic effect, paclitaxel was shown to trigger proinflammatory response by activation of innate immunity, providing an opportunity to explore its combination with immune checkpoint inhibitors 26 . Interestingly, rather than inducing mitotic delay, low doses paclitaxel promote chromosome missegregation and micronucleation which stimulates innate immunity response and promotes antitumor immune surveillance 27 .
We found that addition of the apoptosis potentiator Navitoclax to p31 comet depleted cells accelerated postmitotic death. Our results are in line with previous reports demonstrating that Navitoclax plus antimitotics co-treatment potentiated cancer cell death 28,29 . Surprisingly, although some studies have shown that Navitoclax www.nature.com/scientificreports/ in combination with MTAs prompted mitotic death by accelerating apoptosis in the mitotic-arrested cells 28,29 , we demonstrated that Navitoclax shifted cell death from DiM to PMD in p31 comet depleted cells. This result is in line with another study that showed that inhibition of Bcl-xL by the BH3-mimetic WEHI-539 induces PMD in RKO cells in the presence of paclitaxel 24 . We also found that Navitoclax promotes post-mitotic death of p31 comet -depleted cells after a short delay in mitosis. This suggests that cells that exit mitosis after a delay, here caused by p31 comet knockdown, are committed to die due to Navitoclax-mediated inhibition of antiapototic proteins. Indeed, Bcl-xL (a Navitoclax target) was shown to be crucial for cell survival following an abnormal mitosis 24 . Intriguingly, Navitoclax treatment alone resulted in a strong cytotoxic response (Fig. 5c). However, Navitoclax showed limited single-agent activity in a completed phase II study, and current studies are focusing on combination therapies 30,31 . In this perspective, we believe that its use to enhance/accelerate apoptotic signal accumulation in cancer cells treated with antimitotics could be beneficial by avoiding slippage from mitotic arrest. The p31 comet knockdown/Navitoclax combination could provide maximal conditions for maximal cytotoxicity. Indeed, mechanistically, the combination is much more aggressive in that it promotes rapid cell death, already at the first mitosis, while most of cell killing occurred at mitosis of the second cell cycle in individual treatments (Fig. 5d). This is corroborated by the enhanced reduction of cell survival (Fig. 5a), and could be clinically relevant as it may offer higher efficacy while reducing repeated administration. p31 comet overexpression was previously associated with the abolishment of SAC-dependent mitotic arrest and subsequent mitotic slippage 2,32,33 , as well as with an increased resistance to apoptosis and to antimitotic drugs, such as paclitaxel in cancer cells 32 . Furthermore, mRNA screenings published in oncomine database (www. oncom ine. org) suggested that p31 comet is overexpressed in several cases of lung cancer as well as in other cancers. In line with those evidences, we confirmed that p31 comet was overexpressed at mRNA and protein level in three NSCLC cell lines when compared with a non-tumoral cell line, thus highlighting its potential value as a target for NSCLC therapy.
In conclusion, our data suggest that targeting SAC silencing components, such as p31 comet , can provide a mean to block mitosis and, at the same time, to delay mitotic slippage, thereby providing maximal conditions to enhance cytotoxicity of microtubule poisons and apoptosis-promoting agents. Therefore, targeting SAC silencing can provide a rationale for combination chemotherapy against cancer that deserves to be further explored in a goal to overcome problems of resistance and side effects.

Materials and methods
Cell lines and culture conditions. Cells were grown and maintained as described 34 . NCI-H460 (human non-small cell lung cancer) cells were grown in RPMI-1640 culture medium (Lonza, Basel, Switzerland) with 5% FBS. HPAEpiC (human pulmonary alveolar epithelial cells), A549 (human adenocarcinoma alveolar basal epithelial), and Calu-3 (human lung adenocarcinoma) cells were grown in DMEM medium with 10% fetal bovine serum (FBS, Biochrom) and 1% non-essential amino acids (Sigma Aldrich Co., Saint Louis, MO, USA). Cells were maintained in a 5% CO 2 humidified incubator, at 37 °C. The experiments were performed when cells were at exponentially growing and presented more than 95% viability. The NCI-H460, A549, and Calu-3 cell lines were obtained from American Type Culture Collection. HPAEpiC cell line was purchased from ScienCell Research Laboratories. -mitotic death (green) and death in mitosis (red) over the total number of cells. Cells were transfected with control or p31 comet siRNAs for 48 h, then paclitaxel was added for 24 h, and cells imaged by time-lapse microscopy for 72 h. *p < 0.05, ***p < 0.001, sip31 comet or sip31 comet + paclitaxel vs Control siRNA; # p < 0.05, sip31 comet + paclitaxel vs sip31 comet , ¥¥ p < 0.01, sip31 comet + paclitaxel vs paclitaxel, by two-way ANOVA with Tukey's multiple comparisons test. Cell extracts and Western blotting. Preparation of total cell protein extracts was performed as previously described 35 . For Western Blot analysis, samples were separated by molecular weight using SDS-PAGE gels and transferred to a nitrocellulose membrane. The membrane was blocked with 0.05% Tween-20 with 5% w/v nonfat dry milk and the incubation with antibodies was performed within the same solution. The signal was detected using ECL detection of the HRP-conjugated secondary antibodies. Blots were visualized using X-ray films. Images of X-ray films were captured using Carestream BIOMAX Light Film (Sigma-Aldrich) and quantified by densitometry using ImageJ 1.4v software (http:// rsb. info. nih. gov/ ij/). The primary antibodies were used as follow: rabbit anti-p31 comet (abcam) and mouse anti-α-tubulin (Sigma-Aldrich), diluted at 1/1000 and 1:5000, respectively. Horseradish peroxidase (HRP)-conjugated secondary antibodies were diluted at 1:4000 (anti-mouse, Sigma-Aldrich) or at 1:1000 (anti-rabbit, Sigma-Aldrich). ImageJ 1.4v software was used for the quantification of the intensity of the protein signal. α-Tubulin expression levels were used for normalization.
Mitotic index determination. NCI-H460 cells were seeded in 6-well plates containing complete culture medium at density of 0.1275 × 10 6 cells per well. Cells were counted 48 h after transfection with control-or p31 comet siRNA, or 24 h after paclitaxel treatment. For the p31 comet siRNA and paclitaxel cotreatment, paclitaxel was added 24 h after siRNA transfection. Cells were counted from random microscope fields (n > 2000 for each condition). Round-shaped mitotic cells were quantified over the total cell population for the determination of the mitotic index. Paclitaxel (Sigma-Aldrich) was used at a clinically relevant concentration of 10 nM.  TUNEL assay. In order to detect DNA breaks, Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay was performed. Briefly, cells were plated and treated as above in six-well plates containing coverslips. Coverslips-attached cells were processed with DeadEnd Fluorometric TUNEL System (Promega, Madison, WI, USA), according to the manufacturer's instructions. For DNA staining, 2 µg/ml of DAPI was used in Vectashield mounting medium. TUNEL-positive cells were scored in a total of 500 cells, from at least ten random microscopic fields, under fluorescence microscope, in order to determine the levels of cells undergoing cell death.
Live cell imaging. For live-cell imaging experiments, 5.5 × 10 6 NCI-H460 cells were seeded onto LabTek II chambered cover glass (Nunc, Penfield, NY, USA). Cells were allowed to attach for 24 h at 37 °C with 5% CO 2 , and then transfected with control or p31 comet siRNA or treated with 10 nM of paclitaxel, or 3.5 µM of Navitoclax. For cotreatment, paclitaxel or Navitoclax were added 24 h after siRNA-transfection. Time-lapse imaging was performed 24 h after siRNA transfection or immediately after the addition of paclitaxel or Navitoclax. RPMI without phenol red supplemented with 5% FBS was used in the experiments. Image capture was performed up to 72 h at intervals of 10 min under differential interference contrast (DIC) optics, with a 63× objective. An Axio Observer Z.1 SD inverted microscope (Carl Zeiss, Germany) coupled with an incubation chamber with the temperature set to 37 °C and an atmosphere of 5% CO 2 was used in the experiments. ImageJ software (version 1.44, Rasband, W.S., ImageJ, US National Institutes of Health, Bethesda, MD, USA) was used to produce movies from the images captured during time-lapse imaging. The cells were followed through the entire imaging period and cell fates were tracked for each experimental condition. The number of cells were scored for mitosis and cell death with basis on changes in cell morphology by DIC imaging. Cell death was characterized by cell retraction and plasma membrane blebbing, and mitotic entry by cell rounding. Dead cells were categorized into death in mitosis (DiM) or post-mitotic death (PMD) when death occurred before or following cell division, respectively. www.nature.com/scientificreports/