Janus face-like effects of Aurora B inhibition: antitumoral mode of action versus induction of aneuploid progeny
Janus face-like effects of Aurora B inhibition: antitumoral mode of action versus induction of aneuploid progeny
Ralf Wiedemuth 2
Barbara Klin k 0 1
Mamoru Fujiwara 3
Evelin Schröck 0 1
Masaaki Tatsuka 3
Gabriele Schacker 2
Achim Temme 0 2
0 G,erman Cancer Consortium (DKTK), partner site Dresden, German Cancer Research Center (DKFZ) , 69121 Heidelberg , Germany an
1 I,nstitute for Clinical Genetics, Medical Faculty Carl Gustav Carus, TU Dresden , Fetscherstr. 74, 01307 Dresden , Germany
2 Department of Neurosurgery, Section of Experimental Neurosurgery/Tumor Immunology, University Hospital Carl Gustav Carus, TU Dresden , Fetscherstr. 74, 01307 Dresden , Germany
3 dDepartment of Life Sciences, Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima , Shoubara, Hiroshima 772-0023 , Japan
The mitotic Aurora B kinase is overexpressed in tumors and various inhibitors for Aurora B are currently under clinical assessments. However, when considering Aurora B kinase inhibitors as anticancer drugs, their mode of action and the role of p53 status as a possible predictive factor for response still needs to be investigated. In this study, we analyzed the effects of selective Aurora B inhibition using AZD1152-HQPA/Barasertib (AZD1152) on HCT116 cells, U87-MG, corresponding isogenic p53-deficient cells and a primary glioblastoma cell line. AZD1152 treatment caused polyploidy and non-apoptotic cell death in all cell lines irrespective of p53 status and was accompanied by poly-merotelic kinetochore-microtubule attachments and DNA damage. In p53 wild-type cells a DNA damage response induced an inefficient pseudo-1Gcell cycle arrest, which was not able to halt ongoing endoreplication of cells. Of note, release of tumor cells from AZD1152 resulted in recovery of aneuploid progenies bearing numerical and structural chromosomal aberrations. Yet, AZD1152 treatment enhanced death receptor TRAIL-R2 levels in all tumor cell lines investigated. A concomitant increase of the activating natural killer (NK) cell ligand MIC A/B in p53-deficient cells and an induction of FAS/CD95 in cells containing p53 rendered AZD1152-treated cells more susceptible for NK-cell-mediated lysOisu.r study mechanistically explains a p53-independent mode of action of a chemical Aurora B inhibitor and suggests a potential triggering of antitumoral immune responses, following polyploidization of tumor cells, which might constrain recovery of aneuploid tumor cells.
Members of the aurora kinase family are critically involved In line with this, genetic variants of Aurora A, such as F31I and
in the segregation of sister chromatids and include three -ser T+91A, have been found to be associated with increased cancer
ine/threonine kinases A(URKA/Aurora A, AURKB/Aurora B and susceptibility in a variety of tissues5,(6).
AURKC/Aurora C) (1–3). Despite their similarity at the sequence Aurora B represents a chromosomal passenger protein
level, the localization and functions of Aurora kinases are non- which assembles in a stable complex with the inner cent-ro
overlapping. Aurora A is localized at the centrosome from the meric protein (INCENP/INCENP), BIRC5/Survivin and CDCA8/
time of centrosome duplication through to mitotic exit and r-eg Borealin to build the chromosomal passenger complex (CPC)2().
ulates centrosome function. Aurora A is commonly amplified in INCENP, Survivin and Borealin act as scaffold for Aurora B and
several solid tumors and has been suggested as an oncogen4e).( have regulatory and targeting functions for the CP2)C. A(urora
to elucidate whether AZD1152-induced mitotic defects and
polyploidy define a point of no return and ultimately cause c-an
cer cell death.
Materials and methods
Cell culture and clonogenic survival
The near tetraploid primary glioblastoma cell line HT7606 as well as
U87MGshp53 and HCT116p53−/− cells have recently been described25(
and HCT116 cell lines were authenticated using single-nucleotide p-oly
morphism profiling (Multiplexion GmbH, Heidelberg, Germany). In add-i
tion spectral karyotyping (SKY) was used for HCT116 and HT7606 cell
lines. All cell lines in this study were tested negative for Aurora C- pro
B kinase activity is critically involved in correcting syntelictein expression (data not shown). Long-term survival of AZD1152-treated
and merotelic microtubule–kinetochore (MT–KT) connections U87-MG, HCT116, HT7606 cells and their isogenetic but p53-deficient
counterparts (U87-M Gshp53, HCT116p53−/−) (
) was tested by plating dupl-i
and therefore guarantees biorientation of sister chromatids to cates of 1000 cells per dish onto 1c0m dishes. After 3 weeks, cells were
opposing spindle poles before onset of anaphase (7,8). Together stained with Giemsa and the number of clones was quantified. At least
with its essential role in the execution of cytokinesis, Aurora B, two independent experiments were performed for each cell line. For
in cooperation with its partners of the CPC, safeguards segre-ga Aurora B kinase inhibition cells were either incubated with medium c-on
tion and chromosomal integrity2(). Aurora C has similar stru-c taining 500nM AZD1152-HQPA (Selleckchem) or adequate dimethyl su-l
tural and functional properties as Aurora B but is restricted tofoxide (DMSO) concentrations. To release cells from Aurora B inhibition,
mammalian spermatogenesis ( 9). Aurora B overexpression has cells were washed twice with phosphate-buffered saline (PBS) and further
been reported for several solid cancer1s0(–14). It can be hypoth- cultured in appropriate standard medium.
esized that upregulated Aurora B levels promote tumorigenesis
through induction of tetraploidy and gradual developing a-ne Chromium release assay
uploidy. In line with this notion, we recently demonstrated The ability of primary human natural killer (NK) cells to kill
AZD1152that ectopic overexpression of Aurora B leads to an increase treated tumor cells was analyzed in51aCr release assay as described pr-e
in the fraction of U87-MG glioblastoma cells displaying mitotic viously (
). The use of human NK cells was approved by the local ethical
defects. This effect was further augmented by p53 knockdown committee (#EK242102007). Human peripheral blood mononuclear cells
(15). A mechanism how increased Aurora B levels contribute to (wDerreesdiseonl,aGteerdmeainthy)erorfrforommbfurfefsyhcbolaotosdsoufpphleieadltbhyytdhoenoGresrmafatnerRoebdtCarinoisnsg
tumor progression was recently addressed by Muñoz-Barrera oral and written consent. Target cells× 1(206) were labeled with 50 mCi
et al. (
). By using yeast model systems they corroborated that of 51Cr (PerkinElmer Life Sciences) for 1h at 37°C and then washed four
increased Aurora B activity led to continuous disruption of times with PBS. Labeled target cells were plated as triplicates in
roundamphitelic KT–MT attachments, which ultimately caused s-is bottom 96-well plates at×3103 cells per well and incubated with NK eff-ec
ter chromatin segregation defects. Accordingly, a recent study tor cells at different effector to target (E:T) ratios hf o.Trh6e released51Cr
using a tetracycline-inducible system for expression of Aurora was determined in a beta counter (PerkinElmer Life Sciences). The specific
B showed that induced overexpression of Aurora B resulted in cytotoxicity was calculated as described recent2l5y).(Experiments were
development of aneuploid tumors in mice (
). Intriguingly, no performed at least three times with similar results. Statistical analysis
Aurora B polymorphism which increases cancer susceptibility was performed with Student’st-test.
has been described so far.
It is consistently accepted that Aurora B inhibition using Indirect immunofluorescence analysis
RNA interference, expression of a kinase-inactive, dominant- Cells were stained as described previously26(). Antibodies include mouse
negative mutant of Aurora B1(8), use of soluble circular peptide anti-α-tubulin (1:1000; 1.5h; Sigma), human anti-centromere antibody
IN-box fragments to relocate Aurora B from the CPC19(), and (1:500, kindly provided by K. Conrad, TU Dresden, Germany), polyclonal
chemical Aurora B inhibitors, result in defective mitosis, po-ly isshoetehpioacnytain-maoteus(eu-nCdyi3lu(t1e:1d0;03;m01inh;; ADKiaLnIDoEvSa)AaNnAd palnuts;i-Mhuemdiapnan-f)l.uSotraeisnceedin
ploidy and eventually mitotic catastroph2e0(). Yet, the mecha- cells were imaged with a Leica SP5 inverse microscope (Leica, Wetzlar,
nism leading to the induction of a post-mitotic cell cycle arrest Germany) using 405, 488, 543, 594nm lasers and 63× NA1.4 or 40× NA1.25
remains puzzling (
). Especially, when considering selective objective lenses. Image acquisition, shuttezr-,axis position, laser lines and
Aurora B kinase inhibitors as anticancer drugs, their mode of confocal system were all controlled by Leica LAS AF software. Series in
action and the role of p53 status as a possible predictive factor z-directions z(-stacks) of single cells were obtained at 0.3 µm steps. For
for response have not been fully elucidated. Mechanistically, a analysis of merotelic attachments after h72treatment with AZD1152, the
proposed p53-dependent ‘tetraploidy checkpoint’ which senses acquired images were processed including background subtraction, c-on
increased chromosome numbers and which prevents S-phase trast enhancement, smoothing and 3D reconstruction. A kinetochore was
entry in cells experiencing defective cytokinesi2s3() might limit sspcionrdeldeafisbebresinegmamnearotitneglifcrowmhdeniffietrecnletaprollyescoinnnaze-scttaecdktowroreocromnsotrreuvcitseibdle
the anticancer effectiveness of Aurora B inhibitors by keeping 3D image.
tumor cells in a non-proliferative state. It is also possible that
a p53-dependent cell cycle arrest is due to DNA damage or c-el Western blot analysis
lular stress after tetraploidization or polyploidization. Likewise,
if and how polyploid tumor cells can recover from fully Aurora B
inhibition has not been investigated so far.
Cells lysates were prepared, subjected to electrophoresis and blotted
onto polyvinylidene difluoride membranes as described previously26().
Membranes were probed with primary antibodies [monoclonal rabbit
By using the selective Aurora B inhibitor AZD1152 (Barasertib) p21 waf/cip (Cell Signaling), polyclonal goat anti-p53 (R&D Systems), monoc-lo
), we aimed to mechanistically link the mode of action of nal rabbit anti-p53 S15 (Abcam), polyclonal rabbit anti-p53 S20, polyclonal
Aurora B inhibitors to the p53 status of tumor cells and ind-uc rabbit anti-p53 S37 (Cell Signaling), monoclonal mouse anαt-i-tubulin
tion of a post-mitotic cell cycle arrest. Furthermore, we sought (Sigma), monoclonal rabbit anti-ATM S1981 (Epitomics), polyclonal rabbit
anti-ATM (Merck), monoclonal rabbit anti-caspase 3 (Cell Signaling), mo-no FISH and SKY analysis
clonal rabbit anti-CHK2 T68 (Cell Signaling), polyclonal rabbit anti-cyclin D1For preparation of metaphase chromosomes and interphase nuclei, cells
(Santa Cruz), monoclonal mouse antiγ-H2AX (Millipore), polyclonal rabbit were treated with colcemid for 6m0in at a concentration of 0.035 µg/ml,
anti-histone H3 S10 (Cell Signaling) and monoclonal mouse anti-histone incubated in 0.075M KCl for 20min at 37°C and fixed in a freshly
preH3 (Cell Signaling)] followed by anh1incubation with appropriate second- pared mixture of methanol/acetic acid (3:1) at room temperature. Cell
ary antibodies conjugated with horseradish peroxidase (Dako) at room suspension was dropped onto glass slides. Fluorescencein situ
hybridizatemperature. Signal detection was carried out as described previous2l6y).( tion (FISH) analysis was performed using a commercial probe set targ-et
ing chromosomes X, Y, 13, 18, 21 (XA AneuScore Test-Kit, MetaSystems).
Flow cytometry analysis and cell sorting Signals were counted in at least 150 interphase nuclei per sample and
Staining was carried out in a MACSQuant flow cytometer as described proportions of aneuploid cellsn( signal ≠ 2 or ≠ 4) were compared using
). For analysis of stress-induced surface markers, Pearson’s chi-squared test. SKY analysis was performed as described pr-e
cells were processed and simultaneously stained with monoclonal viously (26).
mouse anti-CD262-PE (TRAIL-R2 DR5, eBioscience), monoclonal mouse
anti-CD95-fluorescein isothiocyanate (FasR, Miltenyi) and monoclonal Results
mouse anti-MIC A/MIC B-APC (Miltenyi) or appropriate isotype controls
(eBioscience, Miltenyi) in 0.5% bovine serum albumin in PBS + 2mM AZD1152 treatment leads to a p53-independent
ethylenediaminetetraacetic acid forh1at 4°C. Subsequently, cells were development of polyploid cells and causes
nonwwaasshuesdedintoPBexScalunddeadneaaldyzceedl.lsP.rAolplideixupmeriiomdeindets(PwI)er(1e pµegr/fmolr,mMedilatetnlyeia)st apoptotic tumor cell death
three times with similar results. Statistical analysis was performed with Treatment with the Aurora B inhibitor AZD1152-induced
Student’st-test. Sorting of polyploid AZD-treated cells with karyotypes cytokinesis defects in HCT116 cells, U87-MG cells, their -iso
≥8n was accomplished using a fluorescence-activated cell sorting (FACS) genic p53-negative counterparts (HCT11p653−/− and U87-MGshp53
ARIA-sorter (BD Bioscience). Prior sorting, HCT116 and HCT1p1536−/− (
)) as well as in the primary glioblastoma cell line HT7606.
cells were treated for 4h8 with 500nM AZD. Treated cells were stained Continuous inhibition of Aurora B was accompanied by ong-o
with Hoechst 33342 (10 µg/ml) and incubated for 3m0in at 37°C. Sorted ing endoreplication of DNA F(igure 1A). The gradual
developcells were continuously passaged. While splitting, an aliquot o×f1205
cells were stained with 10 µg/ml Hoechst and subjected to DNA an-aly ment of polyploidy in p53-deficient cell lines was significantly
sis (MACSQuant). DNA analysis alone or with a concomitant antibody faster when compared with AZD1152-treated, wild-type
staining, BrdU incorporation and Annexin V staining was performed as cells as depicted for cell fractions with DNA contentsn >fo4r
described previously (
). U87-MG and >8n for HCT116 cells F(igure 1B). Whereas the
increase in DNA contents in AZD1152-treated HCT1p1563−/−, AZD1152 became polyploid and simultaneously expressed
sigU87-MGshp53 cells correlated with cell-line-specific doubling nificantly increased levels of cyclin D1 and pw2a1f/cip, whereas
times, the HCT116 and, U87-MG wild-type cells showed less p53-deficient U87-MGshp53 cells showed increased polyploidy
endoreplication F(igure 1B). Since the wild-type and isogenic yet without substantial signs of a post-mitotic cell cycle
p53-deficient cells showed similar individual doubling times arrest F(igure 2C and D). Strikingly, after AZD1152 treatment
(data not shown) this indicates a p53-dependent effect on cell both U87-MG and U87-MGshp53 cells incorporated BrdU even at
cycle progression after chemical inhibition of Aurora B. Yet, DNA contents of 4n, 8n and higher. Yet, the S-phase entry of
clonogenic survival assays of cells after 3 weeks of cont-inu cells expressing wild-type p53 was reduced when compared
ous AZD1152 treatment revealed no surviving clones in all with p53-deficient cells F(igure 2E and F). When
normalcell lines irrespective of p53 statusFi(gure 1C and data not ized to the BrdU-positive cell fractions in the DMSO controls
shown). However, Aurora B inhibition for 7h2 did not result in it became clear that U87-MG cells with functional p53 were
immediate and massive cell death, since we observed only a attenuated to enter S phase when compared with p53-de-fi
moderate rise in the fraction of dead cells compared with c-or cient cells F(igure 2E). In line with this, the absolute amount
responding DMSO-treated controls as depicted iFnigure 1D. To of BrdU-positive cells was also decreased when
AZD1152investigate a possible induction of apoptosis during 24, 48 and treated, p53-positive tumor cells with a DNA content of n>4
72 h AZD1152 treatments, cell lysates of HCT116 and U87-MG were compared with AZD1152-treated cells with knockdown
cells were analyzed for the appearance of cleaved caspase of p53 ( Figure 2F). This indicates some inhibitory effect of p53
3. As shown in Figure 1E, AZD1152 treatment did not induce and its effector p21waf/cip on S-phase entry, which however was
a caspase-dependent apoptosis. In line with this, Annexin not sufficient to stably arrest cells to prevent endoreplication
V-fluorescein isothiocyanate/PI analysis of HCT1p1563−/− cells (Figure 2C and D). Yet, it remains puzzling how chemical inh-i
treated with AZD1152 for 24, 48 and 7h2 revealed a moderate bition of Aurora B triggers p53 activation.
time-dependent increase in PI-positive and double positive PI/
Annexin V cell fractionsS(upplementary Figure 1, available AZD1152 treatment causes poly-merotelic
at Carcinogenesis Online). Yet, the nearly complete absence kinetochore attachments and DNA breaks, which
of Annexin V-positive HCT116p53−/− cell fractions, indicative activates the DNA damage response pathway
of early apoptosis, suggests the induction of a preferentially Especially deregulated MT–KT attachments, which can occur
necrotic-like cell death mechanisms following inhibition of randomly and are frequently established in polyploid cells,
Aurora B (Supplementary Figure 1, available atCarcinogenesis can lead to chromosomal breaks during mitotic slippage27().
Online). Interestingly, AZD1152-treated HCT116 wild-type Therefore, we investigated MT–KT attachments in
AZD1152cells barely show signs of apoptosis or necrotic-like cell death, treated polyploid HCT116 cells. All cells treated forh 7w2ith
which is likely due to the slower development of cells with -kar AZD1152 contained multiple spindles with supernumerary
yotypes >8n (see Figure 1B). That HCT116 cells were still able centrosomes and multiple spindle fibers emanating from se-v
to execute apoptosis was confirmed by analysis of puromycin- eral spindle poles leading to poly-merotelic MT–KT conn-ec
treated cells. Puromycin treatment resulted in the appearance tions. Such disordered MT–KT attachments were not detected in
of cleaved caspase 3 (Figure 1E) and the presence of cell fra-c DMSO-treated control cellFsig(ure 3A).
tions positive for Annexin V (early apoptosis) and double pos-i Since non-resolved poly-merotelic attachments can cause
tive for Annexin V/PI (late apoptosis/necrosis)S(upplementary chromosomal breaks and subsequently a DNA damage response
Figure 1, available atCarcinogenesis Online). (DDR) (
), we hypothesized that abolished Aurora B kinase
function might be linked to the aforementioned activation of
Chemical inhibition of Aurora B results in a p53 and its downstream effector p21waf/cip. In order to detect DNA
p53/p21waf/cip-mediated pseudo-G1 arrest damage on the molecular level we analyzed the expression of
In western blot analysis it became obvious that wild-type cells γH2AX, a marker for DNA double-strand breaks and the acti-va
accumulated p53 protein at 48–72h after Aurora B inhibitor tion of sensor kinases of DDR.
administration, whereas HCT116p53−/−, U87-MGshp53 and DMSO HCT116 cells exposed to Aurora B inhibitor for 7h2exhibited
controls showed no p53 protein and no increase in p53 pr-o increased phosphorylation at serine 1981 oaftaxia telangiectasia
tein levels, respectivelyF(igure 2A and B). Of note, analysis mutated/ATM kinase and at threonine 68 ofcheckpoint homology
of the steady-state expression level of Aurora B at different kinase 2/CHK2 compared with DMSO control cells, indicating an
time points after AZD1152 treatment revealed decreased p-ro activated DDR. Additionallyɣ,H2AX levels were also elevated
tein levels irrespective of p53 status of the U87-MG cells. In in Aurora B-treated cells but were barely detectable in DMSO
parallel, the AZD1152 concentration used for the experiments controls. ɣH2AX levels increased simultaneously with the
completely abolished phosphorylation of Aurora B substrate development of polyploidy as shown in FACS-assisted anal-y
histone H3 at serine 10 in U87-MG and HCT116 cells, co-n sis of AZD1152-treated HCT116 wild-type and HCT11p653−/− cells
firming a switched off Aurora B kinase activity at all inve-sti (Supplementary Figure 2, available atCarcinogenesis Online). In
gated time points (24, 48 and 72h) (Figure 2A). Interestingly, line with the induction of DDR, western blot analysis of p53
p53 accumulation progressively developed after the loss of phosphorylation sites in Aurora B inhibitor treated HCT116
Aurora B function. The p53 activation was accompanied with cells revealed phosphorylation of p53 at S15, S20 and at po-si
an expression of its transcriptional target pw2a1f/cip in U87-MG tion S37 indicative of activated ATM, CHK2 anPdRKDC/DNA-PK
and HCT116 cells but not in isogenic p53-deficient cells or kinases (
) (Figure 3B). That Aurora B inhibition leads to
DMSO controls, respectivelyF(igure 2A and B). A concomitant activation of the DNA damage sensor kinases independently
increase in the protein expression levels of cyclin D1 pointed from p53 was recapitulated in U87-MG and U87-MGshp53 cells
to a potential G1 cell cycle arrest in p53 wild-type cells -fol (Figure 3C). Taken together, inhibition of Aurora B kinase act-iv
lowing Aurora B inhibitionF(igure 2B). When the observed cell ity leads to erroneous KT–MT connections and DNA damage
cycle arrest was investigated on the single cell level using flowwith consecutive activation of DDR sensor kinases and pho-s
cytometry it became obvious that U87-MG cells treated with phorylation of p53.
Tumor cells can resolve AZD1152-induced Interestingly, clonogenic survival assays demonstrated that
polyploidy and recover with increased aneuploidy individual U87-MG, HCT116 as well as p53-deficient isogenic
Antitumor drugs targeting the cell cycle are dose dependent cell clones survived 24 and 4h8 Aurora B inhibitor treatment
and time dependent. Not only the time-concentration product (Supplementary Figure 3A and B, available atCarcinogenesis
but also the exposure time is an important factor when t-ar Online). Also, clonogenic survival assays using HT7606 p-ri
geting mitotic tumor cells. To investigate whether p53-positive mary glioma cells revealed survival of notable numbers of
and tumor cells with loss of p53 are able to recover from c o-m cell clones after 24 and 4h8 AZD1152 treatment, respectively
plete Aurora B inhibition we tested different treatment times. (Supplementary Figure 3C, available atCarcinogenesis Online).
Therefore, we hypothesized that after removing the Aurora B recapitulated when analyzing cells in bright field microscopy.
inhibitor, cells regained their ability to complete mitosis and All cell lines, irrespective of p53 status, showed a phenotype
cytokinesis even with a karyotype of n4 or larger. In order to with typically multinucleated cells and increased cell sizhe 48
test this hypothesis we cultured HCT116, HCT1 1p563−/−, U87-MG, after AZD1152 treatment, which was progressively lost during
U87-MGshp53 and HT7606 cells in presence of Aurora B inhib-i long-term cell culture (see representative images for HCT116
tor for 48h. After removing AZD1152, cells were propagated for and HCT116p53−/− in Figure 4B). In previous studies we
demonseveral days to weeks and analyzed using bright field micr-os strated that polyploid cells were able to form multiple cleavage
copy and FACS-assisted DNA content analysis. Forty-eight furrows in anaphase and multiple ‘propeller-like’ midbodies
hours after AZD1152 treatment (day 0), all tested cell lines in telophase, suggesting that a complete segregation of po-ly
showed a polyploid phenotype ranging from large fractions ploid genomes into several daughter cells is possible15(
of 8n or 16n populations in HCT116p53−/− cells to 4n popula- Therefore, we hypothesized those AZD1152-induced polyploid
tions in U87-MG or 8n in HT7606 cells F(igure 4A). Almost no cells after regaining the ability to execute cytokinesis, separate
2n cell fractions were detectable in HCT116 and U87-MG cells their chromosomes into multiple daughter cells. In turn this
at this time point. Strikingly, with increasing passage nu-m should result in an increased probability of numeric chrom- o
bers, the cells gradually regained their former state of ploidy. somal aberrations in the surviving cells. To prove the assu m-p
For instance, after 16 and 17 days, respectively, DNA co-n tion that cells with restored ploidy are indeed progenies of
tent of U87-MG, U87-MGshp53 and HT7606 cells became indis- former polyploid cells, we checked for increased aneuploidy
tinguishable from the DMSO controls. This observation was in HCT116 and HT7606 cells at day 26 and passage 5 after
AZD1152 release using FISH analysisF(igure 5; Supplementary DMSO-treated control cells (39.1%P ,< 0.01) (Figure 5D and E;
Figure 4, available atCarcinogenesis Online). AZD1152-treated Supplementary Figure 4, available atCarcinogenesis Online).
HCT116 cells, which usually have a stable near diploid kar-yo To ultimately confirm that aneuploid progeny can arise
type, showed an increased fraction of cells with signals c-on from polyploid tumor cells with DNA contents ≥n8and did not
sistent with tetraploidy (44.5%) as well as significant increased represent for instance outgrowing AZD1152-resistant cells we
numbers of cells with aneuploidy (cells with gains or losses performed FACS of HCT116 and HCT116p53−/− cells treated for
of single chromosomes) when compared with DMSO controls 48 h with AZD1152 and having DNA contents of n8 and higher
(AZD: 29.7%, DMSO: 4.6%, P < 0.01) (Figure 5B; Supplementary (Supplementary Figure 5A, available atCarcinogenesis Online).
Figure 4, available atCarcinogenesis Online). Importantly, this When cultivated for several weeks (passage 3 for HCT116 and
significant increase in aneuploidy after AZD release was also passage 4 for HCT116p53−/− cells), we observed that significant
seen if only the near diploid cell fraction (based on FISH -sig fractions of these polyploid cells regained DNA contents onf. 4
nals) was considered (AZD1152: 30.4% versus DMSO: 4.2%, We also detected the appearance of small cell fraction with
P < 0.01) (Figure 5C). SKY of HCT116 treated and control cells 2n-like karyotype in both cell linesSu( pplementary Figure 5B,
confirmed the FISH results and showed additional structural available atCarcinogenesis Online). Of note, FISH analysis corrob-o
chromosomal aberrations indicative for chromosomal breaks rated that HCT116 and HCT11p653−/− cells have regained n2 and 4n
and non-homologous end joining recombination after tre-at karyotypes after transient AZD1152 treatmenStu(pplementary
ment with the Aurora B inhibitorS(upplementary Figure 4C–E, Figure 5C, available atCarcinogenesis Online). Importantly, tra-n
available atCarcinogenesis Online). A similar effect of transient sient AZD1152 treatment resulted in a significant increase
AZD1152 treatment was seen in HT7606 primary glioblastoma of chromosomal aberrations in 70–75% of cells when
comcells, which showed a higher proportion of cells with signal pared with DMSO-treated, n4-sorted controlsS(upplementary
constellations indicative of aneuploidy (56.3%) compared with Figure 5C and D, available atCarcinogenesis Online).
AZD1152-induced expression of MIC A/B ligand for HT7606, which in response to AZD1152 treatment upregulated
activating lectin NKG2D on NK cells renders tumor CD95, TRAIL-R2 and MIC A/B. Since it appears conceivable that
cells as target for NK cells a rise in MIC A/B as well as enhanced death receptors expr-es
Recently, we and others have shown, that inhibition of Aurora B sion levels increase susceptibility of AZD1152-treated tumor
using AZD1152 or knockdown of Survivin increased expression cells to NK cells, we performed cytotoxicity assays. We revealed
of death receptor TRAIL-R2 on glioma cells25(
). Furthermore, that allogeneiicn vitro expanded and interleukin-2-activated NK
we reported that knockdown of Survivin rendered glioma cells cells showed a strong basal cytotoxic reaction against
DMSOmore susceptible for NK-cell-mediated lysis2(5). In order to ana- treated HT7606 and U87-MG glioma cells, which was sign-ifi
lyze the impact of AZD1152 treatment on NK cell susceptibility cantly augmented at higher effector to target ratios (1:10 to 1:25)
we investigated the expression levels of death receptors CD95, when tumor cells were simultaneously treated with AZD1152
TRAIL-R2 and the activating NK cell ligand MIC A/B at incr-eas (Figure 6B).
ing Aurora B inhibitor exposure times using flow cytometry Discussion
(Figure 6A). Noteworthy, CD95 cell surface expression in U87-MG
and HCT116 cells was strongly depended on p53 wild-type st-a Several small molecule inhibitors of Aurora kinases are c-ur
tus and increased at least 6-fold and 30-fold, respectively ath72 rently in clinical evaluation33(,34). Inactivation of Aurora B
exposure time, whereas isogenic p53-deficient cells displayed using pan-Aurora A/B inhibitors ZM44739 and VX680 as well as
only a moderate increase in CD95. In contrast, surface expr-es a recent study using the Aurora B-selective inhibitor AZD1152
sion levels of MIC A/B were significantly increased and higher leads to polyploidy and cell death35(–38). The main open ques
in HCT116p53−/− and U87-MGshp53 cells at 48 and 72h when com - tions in considering Aurora B kinase inhibitors as promising
pared with isogenic cells containing wild-type p53. Remarkably, anticancer drugs are the possible predictive factors for response
we observed a 5-fold to 14-fold increase in TRAIL-R2 levels in and potential unwanted side effects on the tumor cell popu-la
both, p53 wild-type and p53-deficient U87-MG and HCT116 cells, tion. In particular, the activation and role of p53, which has been
respectively, after 72h exposure to AZD1152. We also analyzed suggested to induce a post-mitotic arrest following inhibition
of Aurora B, has remained nebulous 3(8). It has been hypoth- modifications [for reviewing see ref.4(0)]. Intriguingly, a recent
esized that p53 governs a G1 tetraploidy checkpoint and prevents study failed to confirm decreased steady-state protein levels of
endoreplication 2( 3). Furthermore, a study reported by Gully p53 in mouse embryonic fibroblasts after induced expression of
et al. provided evidence that overexpressed Aurora B physically Aurora B (
). Therefore, if inhibition of Aurora B leads to ac-cu
interacts and inactivates p53 by phosphorylating residues S183, mulating levels of p53 is still a matter of debate.
T211 and S215, which leads to enhanced proteasomal degr-a In our comprehensive analysis of AZD1152-treated cells, we
dation besides the well-establishedmdm2/mdm2-mediated now mechanistically show how p53 is activated after Aurora
ubiquitination and degradation pathway22(,39). Therefore, it B inhibition. We consecutively proved that inhibiting Aurora B
appears conceivable that chemical inhibition of Aurora B, which results in polyploidy facilitating incorrect bipolar MT–KT -con
is accompanied by its enhanced proteasomal degradation2(2), nections during mitosis, chromosome breaks and induction of
in part leads to increased half-life of p53. Yet, an increased half- DNA damage sensor kinases which consecutively phosphorylate
life likely does not assure full activation of p53 and concomitant tumor-suppressor p53. Of note, p53 wild-type cells and p53-def-i
cell cycle arrest, which requires additional posttranslationalcient cells resolved chromosome breaks by non-homologous end
joining. Yet, activation of p53 was accompanied with increased released from AZD1152 treatment can recover and give rise to
levels of CDK inhibitor p21waf/cip, and a pseudo-G1 arrest not capa- aneuploid progenies. This could be of clinical relevance since
ble of inhibiting an ongoing endoreplication when cells were this indicates that slow growing tumors might not fully respond
constantly treated with AZD1152. to standard protocols of Aurora B inhibition and develop pr-og
In contrast, a recent investigation from Kumaerti al. failed enies with increased aneuploidy.Vice versa, selective Aurora
to detect a DDR in U2OS cells treated forh24with the Aurora B inhibition might preferentially target highly proliferative
A/B inhibitor ZM447439 2(1). These contradictory results are tumors.
likely due to technical differences, such as incomplete Aurora
A/B inhibition which avoided development of polyploid cells or Supplementary material
different cell lines used for the experiments. On the other hand,
the experimental data of our study are in accordance with our Supplementary Figures 1–5can be found
athttp://carcin.oxfordprevious results obtained after stable knockdown of Aurora B’s journals.org/
molecular chromosomal passenger protein partner Survivin.
Here, RNA-interference-mediated depletion of Survivin induced Funding
similar erroneous KT–MT connections, DDR and pseudo-G1 Wilhelm Sander-Stiftung (2008.065.1 to A.T.).
arrest 2(6). In addition, this study demonstrated the appearance
of numeric chromosomal aberrations as well as structural c-hro
mosomal alterations after knockdown of Survivin. It is therefore Acknowledgements
conceivable that the pseudo-G1arrest after Aurora B inhibition We thank K. Robel, B. Goldberg, P. Freitag and A. Kaessner for
represents a subordinate chromosomal passenger protein ‘loss- excellent technical assistance. The HCT1p1536−/− cell line was
of-function’ phenotype. kindly provided by B. Vogelstein (Johns Hopkins University,
In line with our study, other studies using AZD1152 reported Baltimore).
inhibition of cancer cells and of tumor growth in xenografted Conflict of Interest Statement: None declared.
mice by induction of mitotic catastrophe 2(2,41–44). In our
ienxgpeArZimDe1n1t5s2 wtreeadtidmnenott,owbhseicrhvecoancttrivaadtiicotns roefcceanstparseepo3rftoslslho--wow References
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