Download iASPP and Chemoresistance in Ovarian Cancers

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iASPP and Chemoresistance in Ovarian Cancers – Effects on
Paclitaxel-Mediated Mitotic Catastrophe
LiLi Jiang1,3, Michelle KY Siu1, Oscar GW Wong1, KarFai Tam2, Xin Lu4, Eric W-F
Lam5, Hextan YS Ngan2, Xiao-Feng Le6, Esther SY Wong1, Lara J Monteiro5, HoiYan
Chan1, Annie NY Cheung1
Authors’ Affiliations: Department of 1Pathology, 2Obstetrics and Gynecology, The
University of Hong Kong, HKSAR, China; 3Department of Pathology, West China
Hospital, Sichuang University, Chengdu, China; 4The Ludwig Institute for Cancer
Research Ltd, University of Oxford, United Kingdom; 5Department of Surgery and
Cancer, Imperial College London, London, United Kingdom;
6
Department of
Experimental Therapeutics, Division of Cancer Medicine, the University of Texas M.
D. Anderson Cancer Center, Houston, Texas, USA.
Corresponding Author: Professor Annie NY Cheung, Department of Pathology,
University of Hong Kong, Room 322, The University Pathology Building, 102
Pokfulam Road, Queen Mary Hospital, Hong Kong, HKSAR, China; e-mail:
[email protected]
Running title: iASPP in ovarian cancers
Keywords: iASPP; chemoresistance; ovarian cancer; paclitaxel; mitotic catastrophe
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Translational relevance
Drug resistance and disease recurrence are important concerns for the treatment of
ovarian cancer. The mechanisms underlying chemosensitivity of ovarian cancer cells
remain elusive. Relapsed ovarian cancer is incurable, and there is no universal
accepted therapeutic regime for patients. In this study, we demonstrate that iASPP is
overexpressed in ovarian cancers, highlighting its relationship with chemoresistance
and prognosis. The data presented in this study supports iASPP as a potent predictor
for paclitaxel resistance. Importantly, the association of iASPP with mitotic
catastrophe provides a more rational therapeutic target, which could allow the future
development of cancer intervention, particularly for recurrent patients.
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Abstract
Purpose: iASPP is a specific regulator of p53-mediated apoptosis. Herein, we
provided the first report on the expression profile of iASPP in ovarian epithelial tumor
and its effect on paclitaxel chemosensitivity.
Experimental design: Expression and amplification status of iASPP was examined in
203 clinical samples and 17 cell lines using immunohistochemistry, quantitative
real-time PCR and immunoblotting, and correlated with clinicopathological
parameters. Changes in proliferation, mitotic catastrophe, apoptosis and underlying
mechanism in ovarian cancer cells of different p53 status following paclitaxel
exposure were also analyzed.
Results: The protein and mRNA expression of iASPP was found to be significantly
increased in ovarian cancer samples and cell lines. High iASPP expression was
significantly associated with clear cell carcinoma subtype (P=0.003), carboplatin and
paclitaxel chemoresistance (P=0.04), shorter overall (P=0.003) and disease-free
(P=0.001) survival. Multivariate analysis confirmed iASPP expression as an
independent prognostic factor. Increased iASPP mRNA expression was significantly
correlated with gene amplification (P=0.023). iASPP overexpression in ovarian cancer
cells conferred resistance to paclitaxel by reducing mitotic catastrophe in a
p53-independent manner via activation of separase, whereas knockdown of iASPP
enhanced paclitaxel-mediated mitotic catastrophe through inactivating separase. Both
securin and cyclin B1/CDK1 complex were involved in regulating separase by iASPP.
Conversely, overexpressed iASPP inhibited apoptosis in a p53-dependent mode.
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Conclusions: Our data demonstrate an association of iASPP overexpression with
gene amplification in ovarian cancer and suggest a role of iASPP in poor patient
outcome and chemoresistance, through blocking mitotic catastrophe. iASPP should be
explored further as a potential prognostic marker and target for chemotherapy.
Introduction
Ovarian cancer is the most lethal gynecologic cancer worldwide because it is
frequently diagnosed at advanced stage and demonstrates high rates of
chemoresistance especially in patients with recurrent diseases (1-3). For decades, a
combination of paclitaxel and carboplatin has been used as the first-line
chemotherapy for ovarian cancer. Although apoptosis is usually regarded as the major
mechanism of cell death in response to paclitaxel (4-7), another form of cell death
known as mitotic catastrophe has been observed in a wide range of human cancers
treated with paclitaxel(5-9). Mitotic catastrophe is a distinctive form of cell death
related to abnormal mitosis, but a generally accepted definition is lacking. The
morphology of cells undergoing mitotic catastrophe is initially characterized by
asymmetric chromosomal condensation and missegregation, followed by formation of
nuclear envelops and multi-nucleated giant cells in later stage (10-14). On the other
hand, cytoplasmic condensation and nuclear pyknosis are hallmarks of apoptosis (12).
Previous reports have provided several hypotheses on paclitaxel resistance, but most
of these studies focused on apoptosis caused by paclitaxel (15, 16). The relationship
between paclitaxel resistance and mitotic catastrophe in cancers is still not fully
4
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understood.
Members of the ASPP (the ankyrin repeat, SH3 domain and proline-rich region
containing protein) family have been recently identified as specific regulators of
p53-mediated apoptosis (17-19). Among them, inhibitory member of the ASPP family
(iASPP, encoded by PPP1R13L in human) is considered to be an oncoprotein and is
overexpressed in breast cancer (20) and acute leukemias (21). Moreover, increased
iASPP confers resistance to apoptosis induced by UV radiation or exposure to
cisplatin in human osteosarcoma cells and breast cancer cells (20). The mechanism by
which iASPP is overexpressed is unclear but is unlikely to be directly due to p53
inhibition (22).
In the present study, we provided the first experimental evidence that iASPP is
upregulated
in
ovarian
cancers
in
relation
to
gene
amplification
and
clinicopathological parameters. This study also highlighted the role of iASPP in
chemoresistance to paclitaxel via its effect on mitotic catastrophe.
Material and Methods
Clinical samples and cell lines. One hundred and forty cases of archival
formalin-fixed paraffin-embedded tissues, including 10 ovarian epithelial benign
tumors (ages, 20~77 years; mean age, 32 years), 19 ovarian epithelial borderline
tumors (ages, 20~46 years; mean age, 30 years), 111 epithelial ovarian cancers (ages,
23~78 years; average age, 50 years) and 63 corresponding metastatic foci of ovarian
cancers (supplementary Table 1), were selected for immunohistochemistry from
5
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Queen Mary Hospital, the University of Hong Kong. Among the patients with cancer,
96 have been treated with chemotherapy. Patients were considered chemoresistant if
they did not achieve complete response or recurred within 6 months from the last
chemotherapy regimen. Patients were considered chemosensitive if they remain in
remission or recurred more than 6 months after the last therapy (23). Paired snap
frozen samples of ovarian cancers and their corresponding normal tissues from
another 36 patients with ovarian cancers were also collected for total RNA and
genomic DNA extractions. These frozen blocks have been histologically reviewed and
microdissected to ensure that the carcinoma constitutes to at least 80% of the samples.
The normal tissues were mainly obtained from the ampulla and fimbria of the
Fallopian tubes of the corresponding patients and have also been histologically
examined to ensure absence of invasive or in situ carcinoma. The collection of such
tissue was approved by the Institutional Ethics Board.
Two immortalized normal human ovarian surface epithelium cell lines
(HOSE6-3 and HOSE11-12) and 20 ovarian cancer cell lines were used. Ovarian
cancer cell lines, SKOV-3 and OVCAR3 were purchased from American Type
Culture Collection (ATCC; Manassas, VA, USA). The two HOSE cell lines and
OVCA 420 were kindly provided by Prof. G.S.W. Tsao (Department of Anatomy, the
University of Hong Kong). OC316, DOV13, ES2, PA-1, TOV21G, TOV112D and
SKOV-TR were generous gifts from Dr. Lawrence XF Le (Division of Cancer
Medicine, University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA).
2008 (CDDP-sensitive) and 2008/C13 (CDDP-resistant) cells were provided by Drs.
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S. B. Howell (University of California at San Diego, La Jolla, CA), S. G. Chaney
(University of North Carolina, Chapel Hill, NC), and Z. H. Siddik (M. D. Anderson
Cancer Center) (24), respectively.
2780S and 2780CP were provided by Prof.
Benjamin B.K. Tsang (Department of Obstetrics and Gynecology, University of
Ottawa, Canada). The PEO1, PEO4, PEO14, PEO23, PEA1 and PEA2 cells were
acquired from the Cell Culture Service, Cancer Research UK (London, UK), where
they were tested and authenticated.
Immunohistochemistry,
immunoblotting
and
immunofluorescence.
Immunohistochemistry was performed using EnVision_Dual Link System (K4061;
Dako, Carpinteria, CA) with standard procedures as previously described (25, 26).
Immunoblotting and immunofluorescence were performed as previously described
(27). Antibodies used in this study were summarized in supplementary Table 2. The
specificity of the anti-iASPP antibody used for this study was illustrated by using
immunoblot in clinical samples (supplementary Figure 1B). Omission or replacement
of the primary antibody with PBS was used as a negative control. For
immunoreactivity evaluation, both intensity and percentage of immunoreactivity of
each section were semi-quantitated. Briefly, the level of intensity was recorded as 0
(negative), 1 (weak), 2 (mild), 3 (moderate), and 4 (strong). The percentage of
positive staining was rated as 0 (<5%), 1 (6% to 25%), 2 (26% to 50%), 3 (51% to
75%), and 4 (>75%). The final immunoreactivity score was determined as a product
of the two parameters, giving the range of 0 to 16.
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RNA and DNA Preparation, Quantitative Real-Time PCR (qPCR). Total RNA was
isolated from frozen tissues using Trizol reagent (Invitrogen) according to the
manufacturer’s instruction. First-strand cDNA was synthesized with oligo-dT primer
and SuperScript ൗ Reverse Transcriptase kit (Invitrogen). Genomic DNA was
extracted using phenol/chloroform. ABI PRISM 7900 Sequence Detection System and
SYBR-green PCR master mix (Applied Biosystems) were used for qPCR. All primers
used in this study are listed in supplementary Table 3. The mRNA expression was
normalized with respect to that of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) and determined by 2-CT method after normalization. The primers to
genomic sequences of iASPP were designed using Primer3 (v.0.4.0) software based
on the sequence of intron 2 of iASPP (Ensemble database). LINE-1 was applied as
reference gene for the evaluation of iASPP copy number (28). Each sample was
verified in duplicate. Ovarian cancer samples scoring two-fold difference from the
corresponding normal counterpart were considered as positive for iASPP gene
amplification.
Plasmids,
transfections
and
paclitaxel
treatment.
pcDNA3.1/V5-His-iASPP
expression plasmid containing human full-length cDNA of iASPP (encoded by
PP1R13L) and pSuper plasmid containing siRNA specific to iASPP were used as
described previously(18). Additional siRNAs specific for iASPP and separase were
obtained from Ambion (USA). OVCA 420 and SKOV-3 cells stably expressing iASPP
8
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were generated and selected using G418 (8mg/ml). Transient transfections were
performed using LipofectamineTM 2000 (Invitrogen) according to manufacturer’s
recommendation. Twenty-four hours after transfection, cells were incubated in 1.7M
paclitaxel (a dose corresponding to the lower area under the curve obtained in
pharmacokinetic studies (9, 29)) for 36 hours (knockdown iASPP) or 48 hours
(overexpressed iASPP).
TdT-mediated dUTP nick end labeling (TUNEL) assay and evaluation of apoptotic
and mitotic catastrophe indices. After transfections and paclitaxel treatments,
TUNEL assay was performed using an In Situ Cell Death Detection kit (Roche
Biochemical, Indianapolis, IN) following the manufacturer’s protocol (30). Apoptotic
and mitotic catastrophe figures were also assessed under fluorescence microscopy.
Apoptotic figures were shown by green fluorescein following TUNEL assay. Mitotic
catastrophe figures were observed by the morphological changes in nuclei (stained
with DAPI) as mentioned above (10-14). More than 1,000 viable cells in each
experiment were examined and the apoptotic and mitotic catastrophe indices were
evaluated as percentages of the viable cells counted. Every assay was run in triplicate.
Statistical analysis. Statistical Package for Social Science 15.0 for windows (SPSS
Inc., Chicago, IL, USA) software was used for statistical analysis as described (25).
Mann-Whitney U and Spearman’s test were used to evaluate the difference and
correlation of different groups. Kaplan-Meier method and Cox’s regression model
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were served to estimate the survival probability and multivariate survival analysis. P
value <0.05 was considered as critical significant.
Results
Overexpression of iASPP in ovarian cancers correlates with poor survival. The
expression of iASPP in ovarian benign and borderline tumors as well as invasive
cancers were evaluated (supplementary Table 1). iASPP protein was predominantly
expressed in the cytoplasm, though focal immunoreactivity could be detected in the
nucleus of six borderline tumors and 21 cancers (Figure 1, A). The subcellular
localization of iASPP was confirmed by immunofluorescence in OVCA 420 cell line
(supplementary Figure 1A).
iASPP staining was diffuse and strong in ovarian cancers, in contrast to the
patchy and moderate staining in borderline tumors and focal and weak staining in
benign tumors. There was statistically significant higher expression of iASPP in
borderline tumors (P<0.001) and ovarian cancers (P<0.001) when compared with
benign tumors. In addition, expression of iASPP in ovarian cancers was also
significantly higher than that in borderline tumors (P=0.009). However, there was no
significant difference between the primary carcinomas and their metastatic foci.
Among the four major histological types of ovarian cancers (serous,
endometrioid, clear cell, and mucinous), high iASPP expression was significantly
associated with clear cell subtype when compared with the other histological types
combined (P=0.023) (Figure 1, B). High iASPP expression (immunoreactivity score ≥
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9) was also found to be correlated with shorter overall survival (P=0.003) and
disease-free survival (P=0.001) (Figure 1, C) (supplementary Table 1). There was no
significant correlation between iASPP immunoactivity and histological grade
(P=0.110) or clinical stage of the cancers (P=0.174).
Gene amplification of iASPP contributes to iASPP overexpression. In a panel of 36
paired clinical frozen ovarian cancers specimens, the mRNA levels of iASPP were
found to be significantly up-regulated in cancers when compared with the
corresponding non-tumor counterparts (P<0.001) by qPCR (Figure 1, D upper left
panel).
Six out of the 12 (50%) ovarian cancer cell lines also showed an up-regulation
of iASPP at mRNA level when compared with HOSE6-3 and HOSE11-12 (Figure 2,
A). iASPP has at least two isoforms, the full-length 828 amino acids (aa) isoform and
the iASPP/RAI (351 aa) isoform, both are effective in inhibiting p53-mediated
apoptosis (20). By immunoblotting, increased expression of at least one of the two
iASPP isoforms was observed in nine out of the 13 (69%) ovarian cancer cell lines
(OVCA 420, OV316, DOV13, SKOV-3, PA-1, 2780S, 2780CP, 2008, and 2008/C13)
(Figure 2, B).
To evaluate the mechanisms underlying up-regulation of iASPP, iASPP DNA
copy number was further evaluated by real time PCR (Figure 1, D, upper right panel).
Indeed, 18 out of 36 (50%) cancer samples showed gene amplification of iASPP when
compared with the corresponding non-tumor counterparts. More than 80% of the
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amplified cases displayed elevated mRNA (Figure 1, D lower panel). Spearman’s rho
analysis demonstrated significant correlation between iASPP amplification and its
mRNA expression (P=0.023).
Elevated iASPP is associated with chemoresistance. Among the 111 patients with
ovarian cancers, 96 received chemotherapy after surgical operation. High levels of
iASPP expression were significantly associated with carboplatin and paclitaxel
chemoresistance (P=0.040) (Figure 2, C) (supplementary Table 1). Multivariable
analysis showed high expression of iASPP, chemoresistance, high histological grade
(poor differentiation) and advanced cancer stage were independent predictors of short
overall survival (95% CI=1.046-4.336, P=0.037; 95% CI=6.797-46.154, P<0.001;
95% CI=1.096-3.066, P=0.021; 95% CI=1.182-2.474, P=0.004, respectively) and
disease-free survival (95% CI=1.001-4.059, P=0.050; 95% CI=2.309-11.932, P<0.001;
95% CI=0.957-2.493, P=0.075; 95% CI=1.170-2.473, P=0.005, respectively).
To further investigate the effect of iASPP on chemotherapy, four pairs of
chemo-sensitive and chemo-resistant ovarian cancer cell lines were examined for
iASPP protein level by immunoblotting. SKOV3-TR is a paclitaxel-resistant
derivative of SKOV3, whereas PEO4, PEO23 and PEA2 are cisplatin-resistant
derivatives of PEO1, PEO14 and PEA1, respectively (31, 32). Three of the
chemo-resistant ovarian cancer cell lines, SKOV3-TR, PEA2 and PEO23, showed
higher protein expression of iASPP as compared with their chemo-sensitive
counterparts (Figure 2, D).
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Mitotic catastrophe is the dominant mode of cell death induced by paclitaxel in
OVCA 420 and SKOV-3 cells. By analysis of nuclear morphology, both OVCA 420
and SKOV-3 cells were found to be sensitive to paclitaxel treatment, and cell death
was mainly in the form of mitotic catastrophe with a time-dependent manner (Figure
3,A). At as early as 6 hours after drug treatment, swelling and condensation of
scattered nuclei occurred. After 24 hours, diffuse asymmetric DNA condensation
appeared. At 48 hours, multi-nucleated giant nuclei with chromosome vesicles were
observed and some of the cells started to detach from the coverslips, indicative of
necrosis-like cytolysis. Meanwhile, parallel increased expression of iASPP was
observed (Figure 3, B). The demonstration of mitotic catastrophe as the dominant
mode of cell death was supported by the concurrent up-regulation of cyclin B1
(Figures 5, 6), which was suggested to be a marker of mitotic catastrophe but not
apoptosis (33).
iASPP is involved in normal cell mitosis and paclitaxel-induced mitotic catastrophe.
To visualize the relationship between iASPP and mitotic process, OVCA 420 (Figure
4, A) and SKOV-3 (Figure 4, B) cells were probed with antibodies against iASPP and
β-tubulin simultaneously. Without paclitaxel treatment (Figure 4, first column), iASPP
was observed to be more commonly expressed in mitotic cells than interphase cells in
both cell lines, suggesting involvement of iASPP in mitosis. After paclitaxel exposure,
intense expression of iASPP was observed in cell undergoing mitotic catastrophe
13
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(Figure 4, second column, arrowhead), which further confirmed that iASPP may play
a critical role in mitotic catastrophe type of cell death induced by paclitaxel. Moreover,
among paclitaxel treated cells expressing exogenous iASPP (Figure 4, third column),
many more interphase cells were found to express iASPP (arrow) when compared
with cells in mitotic catastrophe (arrowhead). In contrast, after iASPP knockdown,
paclitaxel treated cells showed universally low expression of iASPP in both interphase
and mitotic catastrophe cells accompanied by an increased frequency of mitotic
catastrophe index (Figure 4, fourth column, arrowhead). Overexpression of iASPP
seems to help cells escaping mitotic catastrophe by favoring interphase state of cancer
cells. In contrast, knockdown iASPP apparently enhances mitotic catastrophe in both
cell lines.
Overexpressed iASPP prevents paclitaxel-induced mitotic catastrophe in a
p53-independent manner and inhibits apoptosis in p53-dependent mode. To further
elucidate the mechanisms by which iASPP affect response of ovarian cancer cells to
paclitaxel, ovarian cancer cell lines with different p53 status were compared using
TUNEL assay. In OVCA 420, an ovarian cancer cell line known to harbor wild-type
p53, although paclitaxel treatment per se under the dose used in our experiments did
not cause significant increase of apoptosis (Figure 5, A), overexpression of iASPP led
to a reduction of apoptotic index in both DMSO (P=0.046) and paclitaxel-treated
groups (P=0.05). More importantly, increased iASPP expression was significantly
associated with reduction of paclitexel-mediated mitotic catastrophe index (P=0.03).
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The same experiments were performed in two other ovarian cancer cell lines with
dysfunctional p53, SKOV-3 and OVCAR3 (Figure 5, A). SKOV-3 cells possess a
single nucleotide deletion at point 267 (codon 90) of TP53, which blocks p53
expression(34) whereas OVCAR3 cells express mutant p53 protein(35). Concurring
with our findings in OVCA 420, significant reduction of paclitaxel-mediated mitotic
catastrophe indices were found after iASPP overexpression in both cell lines (P=0.016
and 0.005, respectively). However, no significant effects on paclitaxel-mediated
apoptosis were observed in these two cell lines. These results implied that
up-regulated iASPP could prevent mitotic catastrophe induced by paclitaxel in a
p53-independent manner, whilst functional p53 is required for paclitaxel-induced
apoptosis in ovarian cancer.
In addition to ectopic expression studies, OVCA 420 and SKOV-3 cell lines
were also knockdown of iASPP using pSuper plasmid and commercial siRNA
silencing iASPP (Figure 5, B and supplementary Figure 2). Consistent with previous
findings, there were increased levels of paclitaxel-induced mitotic catastrophe after
iASPP silencing in both cell lines (P=0.048 and 0.003, respectively), whereas
significantly enhanced apoptosis was only observed in OVCA 420 cells with or
without paclitaxel treatment (P=0.05 and 0.045, respectively).
iASPP affected the securin/cyclin B1/separase pathway. Physiologically, cell mitosis
is highly regulated by mitotic spindle checkpoint in which separase plays an essential
role. Once activated, separase would cleave downstream target multisubunit complex
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(cohesion) and allow the separation of sister chromatids, leading to mitosis. Under
mitotic catastrophe, the normal function of separase may be abrogated resulting in a
failure of division and multinucleation. By immunoblotting, induction of iASPP
followed by paclitaxel exposure in OVCA 420 cells was associated with up-regulation
of separase auto-cleavage (activation) and down-regulation of securin, CDK1 and
cyclin B1 (Figure 5, C). On the other hand, knockdown of iASPP abrogated the
auto-cleavage of separase and enhanced the protein expression of securin, CDK1 and
cyclin B1 upon paclitaxel treatment (Figure 5, C).
As an attempt to clarify the role of the securin/cyclin B1/separase pathway in
iASPP mediated mitotic catastrophe inhibition, siRNA specific to separase was
transfected into OVCA 420 and SKOV-3 cells stably expressing iASPP (Figure 6, A).
Consistent with previous findings (Figure 5, C), ectopic iASPP could elevate active
form of separase (Figure 6, B). As evidence of a central role of separase in mitosis,
our results showed that knockdown of separase could enhance the mitotic catastrophe
irrespective of paclitaxel treatment. More importantly, knockdown of separase could
block iASPP-mediated inhibition of mitotic catastrophe in both OVCA 420 and
SKOV-3 cells, particularly with paclitaxel treatment. These results suggest that iASPP
confers resistance to paclitaxel by reducing mitotic catastrophe via activation of
separase. However, our results cannot rule out the involvement of other signal
molecules in the inhibitory effect of iASPP on paclitaxel-mediated mitotic catastrophe
The mRNA levels of five mitotic checkpoint molecular components, including
Bub 1, Bub 3, BubR 1, Mad 1 and Mad 2, besides separase, securing and cyclin B1,
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were also examined by qRT-PCR. None of them showed significant alterations in
expression levels (data not shown). Taken together, our results suggest that iASPP
might affect the expression of separase via securin and cyclin B1/CDK1 complex at
the protein level.
Discussion
In this study, we demonstrated upregulation of iASPP expression in ovarian cancers
in vivo and ovarian cancer cell lines at both mRNA and protein levels. Furthermore,
high iASPP expression level was significantly correlated with chemoresistance,
shorter overall survival and disease-free survival. Multivariate analysis indicated that
high iASPP expression could serve as a prognostic indicator that was independent of
clinical stage, grade, histological subtype and chemosensitivity in ovarian cancers.
These observations suggest that iASPP may play a role in ovarian carcinogenesis,
predict the patients’ outcome and are likely to exert an effect on chemosensitivity.
To investigate the mechanism of iASPP up-regulation, the relative copy number
of iASPP (PPP1R13L, chromosome 19q13.3), a gene located within the previously
reported 19q amplicon of ovarian cancers, was determined (36). Our data revealed
that half of the cancers demonstrated gene amplification of iASPP, and increased
mRNA expression was significantly correlated with its gene amplification. This is the
first report on iASPP gene amplification in human cancers.
We next sought to investigate the effect of iASPP on chemotherapy in vitro.
Concurring with previous reports on other human malignancies (36), we demonstrated
17
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that paclitaxel could predominantly induce mitotic catastrophes and to a relatively
lesser extent, apoptosis in ovarian cancer cells (OVCA 420 and SKOV-3). One of the
most striking findings in the present study is that overexpressed iASPP could abrogate
not only paclitaxel-mediated apoptosis, but also an alternative mode of cell death,
paclitaxel-mediated mitotic catastrophe. Conversely, knockdown of iASPP could
enhance the mitotic catastrophe.
Unlike apoptosis, the process of mitotic catastrophe is still controversial. Two
mechanisms have been considered to be crucial for mitotic catastrophe, namely G2/M
checkpoint and mitotic spindle checkpoint (38). For G2/M checkpoint, abolishment of
genes such as p53, p21Cip1 and 14-3-3Sigma (39,40) were found to promote DNA
damage-induced mitotic catastrophe. It was therefore logical to speculate that iASPP
may prevent paclitaxel-mediated mitotic catastrophe via p53 (20, 41). But our
findings on OVCAR3 and SKOV-3, two cell lines with dysfunction and deletion of
p53 respectively, that iASPP could still inhibit mitotic catastrophe suggest otherwise.
However, further investigation is needed to understand how this p53 independent
mitotic catastrophe inhibition was mediated. Nevertheless, our work does provide the
first experimental evidence that iASPP plays a pivotal role in mitotic catastrophe in a
p53-independent manner.
Separase is a cysteine protease and regarded as a central downstream target of
mitotic spindle checkpoint (42). The proteolytic activity of separase is regulated by at
least two mechanisms (43). Securin has long been appreciated as a direct inhibitor of
separase, and cell division cycle 2 (also known as CDK1)/cyclin B1 complex
18
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constituted the second pathway via inhibitory phosphorylations. Our data
demonstrated that increased iASPP could enhance the activity of separase via
degradation of securin and cyclin B1/CDK1 complex. As a result, decreased
frequency of paclitaxel-mediated mitotic catastrophe occurred. Conversely, iASPP
suppression enhanced paclitaxel-mediated mitotic catastrophe index following
elevated levels of securin and cyclin B1/CDK1 complex and inactive separase.
Furthermore, siRNA silencing of separase was found to promote the mitotic
catastrophe, which was overcome by overexpressed iASPP. However, we have only
begun to understand the function of iASPP, further research on this versatile protein
and its interaction with separase, securin and/or cyclin B1 is necessary.
On the other hand, recent studies demonstrated the contribution of drug
transporters as important mechanisms of chemoresistance in ovarian cancer (44, 45).
For example, as a primary efflux transporter, P-glycoprotein-mediated drug efflux
could contribute to chemotherapy failure (45). To our best knowledge, there has been
no report about the relationship between iASPP and drug transporters. Further
delineation of the mechanisms would be examined in our future studies.
In summary, iASPP was found to exert a powerful effect on paclitaxel
resistance in ovarian cancers, since it could inhibit both mitotic catastrophe and
apoptosis irrespective of p53 function. Our findings highlight the great potential of
iASPP to be regarded as a biomarker for paclitaxel chemosensitivity and clinical
prognosis in ovarian cancer, as well as an effective chemotherapeutic target.
19
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Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
The authors would like to thank Dr. Wilson Ching for his valuable comments. This
study was supported by funding from the Research Grants Council of the Hong Kong
Special Administrative Region (HKU 7503/06M) and Anti-Cancer Society Fund.
20
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Legends
Figure 1. (A) Representative photomicrograph of iASPP immunoreactivity and (B)
immunoscores of iASPP in benign (b), borderline (bl), and clear cell (c)/endometrioid
(e)/mucinous (m)/serous (s) adenocarcinoma. Scale bar=100μm. Magnified images of
overexpression of iASPP were added in insets. (C) Overall and disease-free survival
curves with respect to iASPP expression. (D) iASPP mRNA expression in clinical
samples (upper left panel) and gene amplification status (upper right panel) in relation
to mRNA expression (lower panel).
Figure 2. (A) mRNA and (B) protein expression pattern of iASPP in HOSE cell lines
and ovarian cancer cell lines (C) Higher iASPP expression was found in the
chemoresistant patients than in the chemosensitive patients. (D) iASPP protein
expression in four pairs of chemo-sensitive and resistant ovarian cancer cell lines
were compared with higher expression demonstrated in chemoresistant cell lines
(PEA2, PEO23). All immunoblot assays were done in duplicate.
Figure 3.
Paclitaxel treatment induced mitotic catastrophe in OVCA 420 and
SKOV-3 cells (A) and parallel increased iASPP (B). OVCA 420 and SKOV-3 treated
with paclitaxel for indicated duration were stained with DAPI and their nuclear
morphology was examined under a fluorescence microscope (arrowhead: asymmetric
chromosomal condensation; arrow: multi-nucleated giant cells). Scale bar=50μm.
24
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Figure 4. Representative pictures of double immunofluorescence images showing
iASPP and -tubulin in (A) OVCA 420 and (B) SKOV-3 cells (control,
vector-transfected control cells; arrow, interphase cells; arrowhead, mitotic
catastrophe). Scale bar=50μm.
Figure 5. Paclitaxel mediated mitotic catastrophe and apoptosis (TUNEL assay) after
(A) ectopic expression of iASPP in OVCA 420, SKOV-3 and OVCAR3 cells and (B)
silencing of iASPP in OVCA 420 and SKOV-3 cells (control, vector-transfected
control cells; arrow, apoptosis; arrowhead, mitotic catastrophe). (C) Expression levels
of iASPP and securin / cyclin B1 / separase pathway in OVCA 420 cells were
illustrated. Scale bar=50μm.
Figure 6. (A) TUNEL assay showed the change of mitotic catastrophe and apoptosis
after
silencing
separase
in
cells
stably
overexpressing
iASPP
(control,
vector-transfected stable cells; siControl, negative control of siRNA; arrow, apoptosis;
arrowhead, mitotic catastrophe). Scale bar=100μm. (B) Parallel protein expressions
were shown by immunoblotting.
Supplementary Figure 1. (A) Confocal microscopic image of OVCA 420 cells
stained
with
anti-iASPP
antibody
demonstrated
that
iASPP
protein
was
predominantly expressed in cytoplasm, but small amount could be detected in the
nucleus. Scale bar=50μm. (B) Immunoblot illustrated the specificity of the
25
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anti-iASPP antibody used for this study in clinical samples.
Supplementary Figure 2. Consistent results were found with iASPP knockdown
demonstrating that abrogating iASPP could enhance paclitaxel-mediated mitotic
catastrophe in both ovarian cancer cell lines (control, vector-transfected control cells;
arrow, apoptosis; arrowhead, mitotic catastrophe).
Supplementary Table 1. Association analysis between iASPP expression and the
clinicopathological features of ovarian epithelial tumours.
Supplementary
Table
2.
Primary
antibodies
used
for
immunoblotting,
immunofluorescence, and immunohistochemistry
Supplementary Table 3. Primers used for quantitative real-time PCR
26
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iASPP and Chemoresistance in Ovarian Cancers - Effects on
Paclitaxel-Mediated Mitotic Catastrophe
LiLi Jiang, Michelle K.Y. Siu, Oscar G.W. Wong, et al.
Clin Cancer Res Published OnlineFirst September 16, 2011.
Updated version
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