Inhibition of GSK 3β Activity Is Associated with Excessive EZH2 Expression and Enhanced Tumour Invasion in Nasopharyngeal Carcinoma
et al. (2013) Inhibition of GSK 3b Activity Is Associated with Excessive EZH2 Expression and Enhanced Tumour
Invasion in Nasopharyngeal Carcinoma. PLoS ONE 8(7): e68614. doi:10.1371/journal.pone.0068614
Inhibition of GSK 3b Activity Is Associated with Excessive EZH2 Expression and Enhanced Tumour Invasion in Nasopharyngeal Carcinoma
Renqiang Ma 0
Yi Wei 0
Xiaoming Huang 0
Ran Fu 0
Xi Luo 0
Xiaolin Zhu 0
Wenbin Lei 0
Jugao Fang 0
Huabin Li 0
Weiping Wen 0
Rajeev Samant, University of Alabama at Birmingham, United States of America
0 1 Allergy and Cancer Center, Otorhinolarygology Hospital, The First Affiliated Hospital of Sun Yat-sen University , Guangzhou , China , 2 Department of Otolaryngology, The Second Affiliated Hospital of Sun Yat-sen University , Guangzhou , China , 3 Department of Otolaryngology, Beijing Tongren Hospital, Capital Medical University , Beijing , China
Background: Enhancer of zeste homolog 2 (EZH2) has been shown to contribute to tumour development and/or progression. However, the signalling pathway underlying the regulation of EZH2 in nasopharyngeal carcinoma (NPC) remains unclear. Since EZH2 contains the putative Glycogen synthase kinase 3 beta (GSK3b) phosphorylation motif ADHWDSKNVSCKNC (591) and may act as a possible substrate of GSK-3b, it is possible that inactivation of GSK3b may lead to excessive EZH2 expression in NPC. Method: We first examined the expression of EZH2 and phosphorylated GSK3b (p-GSK3b) by immunohistochemical staining in NPC samples. Then, we evaluated the interaction of GSK3b and EZH2 using immunoprecipitation and immune blot. Moreover, we determined the effect of inhibition of GSK3b activity on EZH2 expression and tumor invasiveness in NPC cell lines in vitro. Finally, we evaluated the invasive properties of NPC cells after knocking down EZH2 expression with EZH2 siRNA. Results: We found that expression of EZH2 correlated with phosphorylated GSK3b (p-GSK3b) at Ser 9 (an inactivated form of GSK3b) in human nasopharyngeal carcinoma (NPC) samples. We also provided evidence that GSK3b is able to interact with EZH2 using immunoprecipitation and immune blot. Furthermore, we found that inhibition of GSK3b activity can lead to upregulation of EZH2 in NPC cell lines in vitro, with enhanced local invasiveness. By knocking down EZH2 expression with EZH2 siRNA, we found that these invasive properties were EZH2 dependent. Conclusion: Our findings indicate that GSK3b inactivation may account for EZH2 overexpression and subsequent tumour progression, and this mechanism might be a potential target for NPC therapy.
Funding: This study is supported by National Nature and Science Grant of China (No. 81072204, 81072224) and Program for New Century Excellent Talents in
University (No. NCET-10-0851), and Young faculty cultivation project of Sun Yat-sen University (10ykpy10). The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Nasopharyngeal carcinoma (NPC) is a highly malignant disease
with a 5-year overall survival rate of approximately 70% and is
one of the most common cancers in southern China.
Epidemiological data suggest that NPC formation is a result of the interplay
between multiple factors, such as genetic susceptibility,
environmental factors, and EpsteinBarr virus (EBV) infection .
Although excellent results have been achieved on NPC
tumourigenesis, the molecular mechanism underlying NPC pathogenesis
and progression has not been fully elucidated . Consequently,
the survival rate for NPC has not significantly improved even with
the use of radiotherapy, radiochemotherapy or targeted
radiotherapy (as adjuvant therapy), and almost 30% to 40% of patients
will develop distant metastasis within 4 years . Therefore, it is
necessary to elucidate the molecular mechanism(s) underlying local
invasion and early distant metastasis of NPC in order to find novel
therapeutic targets and develop new modalities of treatment.
Recently, it has been suggested that enhancer of zeste homolog
2 (EZH2) is involved in the pathogenesis of NPC by promoting the
transformation of immortalised epithelial cells and enhancing cell
proliferation and differentiation [4,5]. EZH2 is a catalytic subunit
of the polycomb-repressive complex 2 (PRC2), which catalyses
trimethylation of histone H3 lysine 27 (H3K27me3). PRC2 may
recruit other polycomb complexes, DNA methyltransferases, and
histone deacetylases, resulting in additional transcriptional
repressive marks and chromatin compaction at key developmental loci
. Overexpression of EZH2 is a marker of advanced and
metastatic disease in many solid tumours, including prostate
cancer and NPC . For example, Tong et al. suggested EZH2
plays a critical role in cell invasion and/or metastasis by repressing
E-cadherin during the development and/or progression of NPC
. In addition, repression of EZH2 by microRNA-26a is related
to the inhibition of NPC cell growth and tumourigenesis .
However, the signalling pathway underlying EZH2 regulation in
NPC remains unclear.
Glycogen synthase kinase 3 beta (GSK3b) is a serine/threonine
protein kinase involved in glycogen metabolism and the Wnt
signalling pathway, which plays important roles in embryonic
development and tumourigenesis . Active GSK3b is able to
phosphorylate substrates, such as b-catenin and Tau, resulting in
ubiquitin-mediated degradation. GSK3b activity can be abrogated
by direct phosphorylation on the Ser9 residue by
phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-activated protein kinase
(MAPK)/p90RSK, or mammalian target of rapamycin/S6K upon
a number of extracellular stimuli, such as insulin, epidermal
growth factor, or fibroblast growth factor . Wnt signalling
inactivates GSK3b through the phosphorylation of the Ser9
residue and prevents it from phosphorylating b-catenin, thus
stabilising b-catenin in the cytoplasm . Whereas overexpression
of GSK3b can induce apoptosis in several cell types, inactivation
of GSK3b has been found to reduce apoptosis. Moreover,
increasing evidence shows that GSK3b plays a critical role in
linking multiple pathways to regulate cellular apoptosis and
tumourigenesis by direct phosphorylation of a broad range of
substrates, including translation factor eIF2B, cyclin D1, c-Jun,
cmyc, NFAT, cyclic AMPresponsive element binding protein,
Tau, and Snail .
Since GSK3b demonstrates a preference for
pre-phosphorylated (primed) substrates by recognising the consensus sequence
S/TX-X-X-Phospho-S/T [10,11] and EZH2 contains the putative
GSK-3b phosphorylation motif ADHWDSKNVSCKNC (591),
EZH2 may be a candidate substrate of GSK3b, and GSK3b
inactivation may lead to excessive EZH2 expression in NPC. To
test this hypothesis, we examined the expression of EZH2 and
pGSK3b (Ser9) in NPC specimens and investigated the possible
regulatory mechanism in vitro. Our findings regarding
GSK3bregulated EZH2 expression may be beneficial for understanding
the pathogenic mechanism of NPC and improve the prognosis of
Materials and Methods
The research protocols were approved by the Ethics Committee
of the First Affiliated Hospital of Sun Yat-sen University. All NPC
and control participants with tissue examination provided their
written informed consent to participate in this study.
Constructs and reagents
The human NPC cell lines CNE-1 and CNE-2 were obtained
from the Cancer Center of Sun Yat-sen University. The
kinasedead GSK3b (GSK3b-KD) and constitutively active GSK3b
(GSK3b-CA) plasmids were kindly provided by Qingqing Ding
from (MD Anderson Cancer Center). Lithium chloride was
obtained from Sigma-Aldrich. Antibodies (rabbit anti-human
GSK3b, rabbit anti-human p-GSK3b (Ser9) and rabbit
antihuman EZH2) were purchased from Cell Signal Technology.
Rabbit anti-human GAPDH was obtained from Santa Cruz
Immunohistochemical and immunofluorescent staining
Immunohistochemical staining was performed as previously
reported . Briefly, human tissue sections were stained for the
expression of phosphorylated GSK3b (Ser9) (1:200) and EZH2
(1:200) and detected by streptavidinbiotinhorseradish
peroxidase complex formation. Immunoglobulin G was used as a
negative control instead of primary antibodies. Two independent
observers blind to the diagnoses and clinical data counted the
number of positive cells in 5 randomly selected high-power fields
(HPFs, 4006), and the numbers were averaged.
Cell culture, transient transfection and RNA interference
The human NPC cell lines CNE-1 and CNE-2 were cultured in
RPMI-1640 supplemented with 10% foetal bovine serum
(Hyclone). Transient transfection with GSK3b-CA or KD plasmid
(2 mg/mL) was performed with Lipofectamine 2000 reagent
(Invitrogen) according to the manufacturers protocol. In addition,
lithium (20 mmol/L) was used to inhibit the activity of GSK3b.
For RNA interference, EZH2 siRNA (50 nmol/L) was transfected
into NPC cells with Lipofectamine 2000 reagent (Invitrogen)
according to the manufacturers protocol.
Immunoprecipitation and immune blot analysis
For western blot analysis, whole cell lysates (KeyGEN) were
resolved by SDS-PAGE, followed by immunoblotting using
Figure 1. Expression of p-GSK3b (Ser9) and EZH2 in NPC tissues and normal controls. (A) Representative immunohistochemical staining
results of EZH2 and p-GSK3b (Ser9) in NPC and control tissues; (B) The mean number of EZH2 positive cells is greater in NPC tissues than in normal
controls; (C) The mean number of p-GSK3b positive cells is greater in NPC tissues than in normal controls; (D) Coincident high or low EZH2 and
pGSK3b (Ser9) immunohistochemical staining in 2 representative NPC tissues; (E) Immunoreactivity of EZH2 is positively associated with p-GSK3b
(Ser9) immunoreactivity in NPC tissues; (F) Immunoreactivity of EZH2 is positively associated with higher stage of NPC.
antibodies at the following dilutions: anti-p-GSK3b (Ser9) (1:500),
anti-GSK3b (1:1000), anti-EZH2 (1:500) and anti-GAPDH
(1:3000). For immunoprecipitation, precleared whole cell lysates
were immunoprecipitated with anti-GSK3b (1:100) insolubilised
on protein G Plus/protein A agarose suspension (CalBiochem).
Then, immunoprecipitates were loaded on a 10% SDS-PAGE and
immunoblotted with different antibodies as described above. Blots
were developed using the ECL detection system
Cell migration and invasion assays
Cell migration was measured using the scratch assay as
described elsewhere . Briefly, CNE-1 and CNE-2 cells were
grown in serum-free medium until 90100% confluency was
reached. After GSK3b-KD or CA plasmid (2 mg/mL) were
transfected for 24 h, a 3-mm wound was introduced across the
diameter of each plate. The scratch area was measured using
ImageJ. The cell covered area was calculated again 48 h after
transfection. Cell invasion was detected by transwell invasion
assay, which was performed as described elsewhere . Briefly,
CNE-1 and CNE-2 cells were grown in serum-free medium until
90100% confluency was reached. The assay was performed using
chambers with an 8 micron pore size polyethylene terephthalate
membrane and a thin layer of matrigel basement membrane
matrix. After GSK3b-KD or CA plasmid (2 mg/mL) was
transfected for 72 h, the cells on the underside of the filter were
fixed, stained and counted.
The number of positive cells in tissues was expressed as the
median and 2575th percentile and analysed using a
nonparametric Mann-Whitney U-test. The Spearman rank correlation test
was used to analyse the correlation among different parameters.
The in vitro data were expressed as the mean and standard error of
the mean (SEM) and analysed using an ANOVA and a two-tailed
t-test. A P-value less than 0.05 was considered statistically
Correlation between GSK3b inactivation and EZH2
expression in NPC tissues and cell lines
Given that EZH2 contains a putative GSK3b phosphorylation
motif, we first tested whether there was a correlation between
EZH2 expression and GSK3b inactivation in NPC specimens. As
shown in Fig 1A, both EZH2 and p-GSK3b (Ser9) protein
expression showed specifically nuclear and cytoplasmic
distribution. To quantify the expression of EZH2 and p-GSK3b (Ser9), we
counted and averaged the number positive cells in 5 randomly
selected HPFs. Consequently, we found the mean number of
EZH2-positive cells per HPF was 35.4 [14.0, 50.2] and 4.8 [2.0,
13.4] in NPC and control tissues, respectively. Similarly, the mean
number of p-GSK3b (Ser9)-positive cells per HPF was 11.2 [7.7,
18.5] and 3.2 [1.0, 5.8], respectively. These results showed that the
levels of p-GSK3b (Ser9) and EZH2 immunoreactivity in NPC
specimens were significantly higher than those in normal
nasopharyngeal tissues (p,0.05 for both) (Fig 1B and C). There
was a significant association between p-GSK3b (Ser9) and EZH2
immunoreactivity in NPC specimens by Spearman rank
correlation (r = 0.75, p,0.05) (Fig 1D and E). In addition, we evaluated
the relationship between EZH2 immunoreactivity and clinical
severity of NPC. We found EZH2 immunoreactivity to be
positively associated with tumour stage (r = 0.89, p,0.05) (Fig 1F).
Evidence for the interaction between GSK3b and EZH2 in
By using molecular structure analysis, we found that EZH2
contains a putative GSK3b phosphorylation motif,
ADHWDSKNVSCKNC (591), and thus, EZH2 may be a
candidate substrate of GSK3b (Fig 2A). To examine whether
GSK3b could interact with EZH2, lysates from CNE-1 and
CNE2 cells were used for GSK3b and EZH2 co-immunoprecipitation.
As shown in Fig 2B, the interaction between GSK3b and EZH2
was clearly detected by western blot analysis.
GSK3b inactivation is associated with EZH2
overproduction in vitro
To investigate whether GSK3b regulates EZH2 expression,
CNE-1 and CNE-2 cells were transfected with GSK3b-CA or KD
plasmids, and EZH2 protein expression was examined by immune
blot analysis. As illustrated in Fig 3, when CNE-1 and CNE-2 cells
were transfected with GSK3b-CA, we observed that both
pGSK3b (Ser9) and EZH2 were significantly downregulated in
CNE-1 and CNE-2 cells. However, when GSK3b activity was
inhibited after cells were transfected with GSK3b-KD or treated
with lithium, both p-GSK3b (Ser9) and EZH2 were significantly
upregulated in CNE-1 and CNE-2 cells. These findings provided
further evidence that excessive EZH2 expression is associated with
the inactivation of GSK3b.
GSK3b inactivation promoted the stability of EZH2
protein in vitro
To investigate the molecular mechanism underlying EZH2
expression after inhibition of GSK-3b activity, we examined
EZH2 mRNA level in CNE-1 and CNE-2 cells after GSK3b-KD
transfection. However, not significant effect of GSK3b
inactivation on EZH2 was observed (data not shown). To further
investigate whether GSK3b exert a posttranscriptional regulation
on EZH2 production, we then examined the stability of EZH2
protein in CNE-1 cells after GSK3b-KD transfection (2 mg/mL)
in vitro. As illustrated in Fig 4, when CNE-1 cells were treated
with cycloheximide (20 mM) for the indicated times after
transfection, we found the half-life of EZH2 protein in
GSK3bKD group was significantly longer than in normal control
(p,0.05). Therefore, our finding showed inhibition of GSK3b
activity can promote EZH2 overproduction by increasing the
stability of EZH2 protein.
Figure 4. GSK3b inactivation promoted the stability of EZH2 protein in vitro. (A, B) Representative western blot analysis of EZH2 after
GSK3b-KD (2 mg/mL) or control plasmid transfection in CNE-1 cells; CNE-1 cells were treated with cycloheximide (20 mM) after transfection, and EZH2
protein level in the indicated time point was detected by western blot analysis which containing equal amounts of protein. (C) The half-life of EZH2,
as suggested by the relative EZH2 intensity, was significantly longer in GSK3b-KD group than in normal control (p,0.05). The data indicate the means
(SEM) of 3 independent experiments. KD: kinase-dead GSK3b plasmid; NC, normal control plasmid.
Figure 5. Inhibition of GSK-3b activity enhanced migration of NPC cell lines. (A) Representative images showing the cell covered area on
the culture plate containing NPC cells after transfection with GSK3b plasmid. Inhibition of GSK-3b by GSK3b-KD transfection enhanced migration of
CNE-1 and CNE-2 cells, whereas activation of GSK-3b by GSK3b-CA transfection suppressed migration of CNE-1 and CNE-2 cells; (B) Quantitative
analyses for the cell covered areas showed that the migrated cells in the GSK3b-KD group increased significantly, whereas those in GSK3b-CA group
decreased significantly when compared to the control. Migration of NPC cells was evaluated by scratch assay after GSK3b-KD or CA plasmid (2 mg/
mL) transfection for 24 or 48 h. The data indicate the means (SEM) of 3 independent experiments. NC: normal control; CA: constitutively active GSK3b
plasmid; KD: kinase-dead GSK3b plasmid.
GSK3b inactivation and EZH2 upregulation is associated
with enhanced invasive capacity of NPC cell lines in vitro
Because EZH2 has been shown to play a critical role in cell
invasion and/or metastasis during the tumourigenesis of NPC, we
investigated whether GSK3b inactivation and subsequent EZH2
upregulation affected the invasion of NPC cells using the cell
scratch assay. As illustrated in Fig 5, after transfection with
GSK3b-KD or GSK3b-CA plasmid for 48 h, we found the
covered area of migrated cells was significantly smaller in the
GSK3b-CA group, where EZH2 was downregulated, but
significantly larger in the GSK3b-KD group, where EZH2 was
upregulated, when compared to the control group. Moreover,
the ability of cells to invade matrigel indicates the invasive capacity
of the CNE-1 and CNE-2 cell lines. By transwell invasion assay,
we found that the number of invaded cells was significantly less in
the GSK3b-CA group and significantly more in the GSK3b-KD
group when compared to the control group (Fig 6). Taken
together, these findings indicate that GSK3b inactivation
enhances the migratory and invasive capacities of NPC cell lines
To further test whether EZH2 was involved in the enhanced
invasion of NPC cell lines followed by GSK3b inactivation, we
transfected EZH2 siRNA into NPC cells to inhibit EZH2
expression under different conditions. As illustrated in Fig 7,
EZH2 siRNA transfection significantly changed the covered area
of migrated cells in the scratch assay, as well as the number of
invaded cells in the transwell assay. The effects of EZH2 siRNA on
the covered area of migrated cells, as well as the number of
invaded cells, were especially significant in the GSK3b-KD group.
These findings suggest that EZH2 is essential for the enhanced
migratory and invasive capacities of NPC cell lines after GSK3b
In the present study, we present the preliminary clinical and in
vitro data suggesting a possible role for GSK3b in the regulation of
EZH2 and subsequent progression of NPC. Our findings suggest
that an aberrant GSK3b/EZH2 regulatory axis may be critical for
initialising the formation of NPC. NPC is known to be a prevalent
malignant neoplasm with a distinct epidemiology and
geographical distribution. Currently, southern China has the highest risk
worldwide, and there are many advanced patients suffering from a
poor prognosis. Although the molecular events responsible for the
progression of NPC remain to be elucidated, the common
mechanism appears to be the aberrant activation of developmental
signalling pathways, leading to uncontrolled cell proliferation. By
examining the mechanism through which GSK3b regulates
excessive EZH2 production, our findings present promising
evidence for developing a potential therapeutic target for the
future management of NPC.
Gene expression is regulated at a number of different levels, one
of which is the accessibility of genes and their controlling elements
to the transcriptional machinery. EZH2 can bind the DNA
methyltransferases DNMT1, DNMT3A, and DNMT3B, which
can result in DNA methylation in certain circumstances .
Although several reports in the literature documented
overexpression of EZH2 and EZH2-dependent tumourigenesis in human
Figure 7. GSK3b-enhanced migration and invasion of NPC cells were abrogated by EZH2 siRNA transfection. (AC) The siRNA
knockdown efficiency was evaluated by testing the protein level of EZH2. EZH2 siRNA transfection significantly reduced EZH2 expression in CNE-1
and CNE-2 cells; (D,E) Inhibition of EZH2 by EZH2 siRNA (50 nmol/L) transfection significantly inhibited migration of CNE-1 and CNE-2; (F,G) EZH2
siRNA (50 nmol/L) transfection significantly inhibited GSK3b-KD-dependent migration of CNE-1 and CNE-2; (H,I) Inhibition of EZH2 by EZH2 siRNA (50
nmol/L) transfection significantly inhibited invasion of CNE-1 and CNE-2; (J,K) EZH2 siRNA transfection significantly inhibited GSK3b-KD-dependent
invasion of CNE-1 and CNE-2. Migration and invasion of NPC cells were evaluated after EZH2 siRNA (50 nmol/L) and/or GSK3b-KD plasmid (2 mg/mL)
were transfected for 48 or 72 h. The data indicate the means (SEM) of 3 independent experiments. NC: normal control; KD: kinase-dead GSK-3b
NPC [4,5,16,17], the precise molecular mechanisms leading to
EZH2 upregulation remain largely unknown. In agreement with
these studies, we observed high EZH2 expression in this group of
NPC specimens. EZH2 expression was positively associated with
clinical severity, suggesting that EZH2 upregulation can
contribute to the local invasion of NPC. Moreover, we found EZH2
expression is significantly related to the inactivation of GSK3b
(Ser9) in these NPC specimens. Since GSK3b demonstrates a
preference for pre-phosphorylated (primed) substrates by
recognising a consensus sequence and EZH2 contains the putative
GSK3b phosphorylation motif ADHWDSKNVSCKNC (591), we
hypothesised that GSK3b may exert a regulatory effect on EZH2
by site-specific phosphorylation. As we suspected, when GSK3b
and EZH2 were co-immunoprecipitated from NPC cell lysates,
the interaction between GSK3b and EZH2 was clearly detected
by immune blot, indicating GSK3b is able to recognise and bind
to EZH2. Due to technical restriction, our working on site-specific
phosphorylation of EZH2 is still in progress, we thus are unable to
show the evidence of phosphorylation of EZH2 in response to
GSK3b in this study. Future data on the specific phosphorylation
site of EZH2 by GSK3b transfection is therefore of great interest.
Recently, GSK3b has become an important area of
investigation as a key component of the Wnt signalling pathway. Unlike
other protein kinase, GSK3b is constitutively active in resting cells
and undergoes a rapid and transient inhibition in response to a
number of external signals . GSK3b activity is regulated by
site-specific phosphorylation as well. Full activity of GSK3b
generally requires phosphorylation at tyrosine 216 (T216), and
conversely, phosphorylation at serine 9 (Ser9) leads to the
inhibition of GSK3b activity . GSK3b also participates in
neoplastic transformation and tumour development. The role of
GSK3b in tumourigenesis and cancer progression remains
controversial; it may function as a tumour suppressor for
certain types of tumours but promotes growth and development
for some others . A variety of signalling pathways may
contribute to NPC carcinogenesis. For example, the EBV-encoded
latent membrane proteins (LMP1, LMP2A, and LMP2B) have
been associated with activation of PI3K/Akt and extracellular
signal-regulated kinase (ERK)/MAPK [20,21], and LMP2A has
been shown to activate the protooncogenic Wnt signalling
pathway . However, there is scant literature addressing the
role of GSK3b in the signalling pathways underlying the
carcinogenesis of NPC. In a previous study, we demonstrated
that GSK3b inactivation is associated with tumour stage of NPC
through regulation of PMS2 . Similarly, Morrison et al.
established the significance of GSK3b inactivation in the
ubiquitin-mediated degradation and stabilisation of b-catenin
production and NPC progression .
In this study, although we were unable to identify the specific
phosphorylation site of EZH2, but the observed interaction of
GSK3b and EZH2 in NPC cells prompted us to further investigate
the regulatory effect of GSK3b on EZH2 production in vitro. For
this reason, we then transfected GSK3b-CA or GSK3b-KD
plasmid or used lithium as a specific inhibitor to regulate GSK3b
activity in cell lines. Since we observed significant change in
halflife of EZH2 protein but not mRNA expression in response to
GSK3b transfection, we concluded that GSK3b may exert its
effect on EZH2 expression in the protein level. When GSK3b
activity was enhanced by transfection with GSK3b-CA, we
observed that active GSK-3b production was significantly
upregulated and EZH2 production was significantly inhibited in
CNE-1 and CNE-2 cells. Moreover, when GSK3b activity was
inhibited upon transfection with GSK3b-KD or lithium treatment,
both p-GSK3b (Ser9) and EZH2 were significantly upregulated in
CNE-1 and CNE-2 cells. This finding suggested there may exist a
balance between activated and inactivated form of GSK3b, and
the mechanism still need further investigation.
Although we did not exclude other pathways that may be
involved in EZH2 overexpression in human NPC tissues, our
finding provided the preliminary evidence that EZH2 expression is
regulated by GSK3b with phosphorylation on Ser9. EZH2
belongs to the family of polycomb group proteins and plays a
master regulatory role in many important cellular processes. There
is increasing evidence that overexpression of the EZH2 gene
occurs in a variety of human malignancies, and abnormalities of
this gene correlate closely with tumour aggressiveness and/or poor
patient prognosis [25,26]. However, the status and function of
EZH2 have not yet been clearly documented in NPC. Recently,
Lu et al. reported that knockdown of EZH2 induced cell growth
inhibition and a G1-phase arrest, and EZH2 overexpression could
rescue the growth suppressive effect in NPC cells .
Furthermore, Tong et al. demonstrated that expression of EZH2 in NPC
cells and nasopharyngeal tissues correlated with
clinicopathological features and survival of NPC patients, and the expression
levels of EZH2 influenced the invasive capacity of NPC cell lines in
vitro . In this study, we also found that inactivation of GSK3b
and subsequent EZH2 overexpression promoted local invasion of
NPC cells. By cell scratch assay, we found migration was
significantly enhanced in the GSK3b-CA group with
downregulated EZH2 but was significantly impaired in the GSK3b-KD
group with upregulated EZH2. Similar effects on cell invasion
were observed in the two groups of NPC cells by transwell invasion
assays. Taken together, these findings clearly indicate the potential
importance of a dysregulated GSK3b/EZH2 axis in the
progression of NPC, which might hold significant promise for identifying
critical molecular targets and improving NPC therapy.
In summary, our findings preliminarily indicate that excessive
EZH2 production in human NPC tissues may result from
inactivation of GSK3b, which was measured by phosphorylated
GSK3b on Ser9 residue. Furthermore, we provide evidence that
GSK3b is able to bind to EZH2 in vitro and that inhibition of
GSK3b activity is associated with excessive EZH2 production,
which may enhance the local invasion capacity of NPC cells.
Therefore, this newly identified mechanism will be helpful to
expand the understanding of NPC tumorigenesis and design
potential therapeutic strategy for NPC future management.
Conceived and designed the experiments: WW HL. Performed the
experiments: RM YW RF XL XH XZ JF. Analyzed the data: YW WL.
Contributed reagents/materials/analysis tools: RM YW. Wrote the paper:
11. Bax B , Carter PS , Lewis C , Guy AR , Bridges A , et al. ( 2001 ) The structure of phosphorylated GSK-3beta complexed with a peptide, FRATtide, that inhibits beta-catenin phosphorylation . Structure 9 : 1143 - 52 .
12. Huang XM , Dai CB , Mou ZL , Wang LJ , Wen WP , et al. ( 2009 ) Overproduction of cyclin D1 is dependent on activated mTORC1 signal in nasopharyngeal carcinoma: implication for therapy . Cancer Lett 279 : 47 - 56 .
13. Liu Y , Zhou Y , Zhu K ( 2012 ) Inhibition of glioma cell lysosome exocytosis inhibits glioma invasion . PLoS One 7 : e45910 .
14. Sun W , Guo MM , Han P , Lin JZ , Liang FY , et al. ( 2012 ) Id-1 and the p65 subunit of NF-?B promote migration of nasopharyngeal carcinoma cells and are correlated with poor prognosis . Carcinogenesis 33 : 810 - 7 .
15. Vire E, Brenner C , Deplus R , Blanchon L , Fraga M , et al. ( 2006 ) The Polycomb group protein EZH2 directly controls DNA methylation . Nature 439 : 871 - 4 .
16. Alajez NM , Shi W , Hui AB , Bruce J , Lenarduzzi M , et al. ( 2010 ) Enhancer of Zeste homolog 2 (EZH2) is overexpressed in recurrent nasopharyngeal carcinoma and is regulated by miR-26a, miR -101, and miR-98. Cell Death Dis 1 : e85 .
17. Hwang CF , Huang HY , Chen CH , Chien CY , Hsu YC , et al. ( 2012 ) Enhancer of zeste homolog 2 overexpression in nasopharyngeal carcinoma: an independent poor prognosticator that enhances cell growth . Int J Radiat Oncol Biol Phys 82 : 597 - 604 .
18. Grimes CA , Jope RS ( 2001 ) The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling . Prog Neurobiol 65 : 391 - 426 .
19. Doble BW , Woodgett JR ( 2003 ) GSK-3: tricks of the trade for a multi-tasking kinase . J Cell Sci 116 : 1175 - 86 .
20. Farago M , Dominguez I , Landesman-Bollag E , Xu X , Rosner A , et al.( 2005 ) Kinase-inactive glycogen synthase kinase 3beta promotes Wnt signaling and mammary tumorigenesis . Cancer Res 65 : 5792 - 801 .
21. Swart R , Ruf IK , Sample J , Longnecker R ( 2000 ) Latent membrane protein 2Amediated effects on the phosphatidylinositol 3-Kinase/Akt pathway . J Virol 74 : 10838 - 45 .
22. Morrison JA , Klingelhutz AJ , Raab-Traub N ( 2003 ) Epstein-Barr virus latent membrane protein 2A activates beta-catenin signaling in epithelial cells . J Virol 77 : 12276 - 84 .
23. Fang J , Lei W , Huang X , Li P , Chen X , et al. ( 2012 ) Expression of mismatch repair gene PMS2 in nasopharyngeal carcinoma and regulation by glycogen synthase kinase-3 in vivo and in vitro . Auris Nasus Larynx 39 : 71 - 6 .
24. Morrison JA , Gulley ML , Pathmanathan R , Raab-Traub N ( 2004 ) Differential signaling pathways are activated in the Epstein-Barr virus-associated malignancies nasopharyngeal carcinoma and Hodgkin lymphoma . Cancer Res 64 : 5251 - 60 .
25. Cao Q , Yu J , Dhanasekaran SM , Kim JH , Mani RS , et al. ( 2008 ) Repression of E-cadherin by the polycomb group protein EZH2 in cancer . Oncogene 27 : 7274 - 84 .
26. Min J , Zaslavsky A , Fedele G , McLaughlin SK , Reczek EE , et al. ( 2010 ) An oncogene-tumor suppressor cascade drives metastatic prostate cancer by coordinately activating Ras and nuclear factor-kappaB . Nat Med 16 : 286 - 94 .