Downregulation of Polo-Like Kinase 4 in Hepatocellular Carcinoma Associates with Poor Prognosis
et al. (2012) Downregulation of Polo-Like Kinase 4 in Hepatocellular Carcinoma Associates with Poor
Prognosis. PLoS ONE 7(7): e41293. doi:10.1371/journal.pone.0041293
Downregulation of Polo-Like Kinase 4 in Hepatocellular Carcinoma Associates with Poor Prognosis
Lili Liu 0
Chris Zhiyi Zhang 0
Muyan Cai 0
Jia Fu 0
George Gong Chen 0
Jingping Yun 0
Shree Ram Singh, National Cancer Institute, United States of America
0 1 State Key Laboratory of Oncology in Southern China, Sun Yat-Sen University Cancer Center , Guangzhou , China , 2 Department of Pathology, Sun Yat-Sen University Cancer Center , Guangzhou , China , 3 Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong , Shatin, N.T. , Hong Kong
Polo-like kinase 4 (PLK4), belonging to serine/threonine kinase family, is critical for centriole replication and cell cycle progression. PLK4 has been proposed as a tumor suppressor in hepatocellular carcinoma (HCC). However, its expression and significance in HCC have not been well studied. In the present study, we found that PLK4 was markedly downregulated in both HCC cell lines and fresh cancer tissues, using quantitative real-time-PCR and western blot. Immunohistochemistry data also revealed that decreased expression of PLK4 was present in 72.4% (178/246) of HCC tissues, compared with the corresponding adjacent nontumorous tissues. Furthermore, PLK4 expression significantly correlated with clinicopathological parameters, including clinical stage (P = 0.034), serum a-fetoprotein (AFP) (P = 0.019) and tumor size (P = 0.032). Moreover, HCC patients with low PLK4 expression survived shorter than those with high PLK4 expression, as indicated by overall survival (P = 0.002) and disease-free survival (P = 0.012) assessed by the Kaplan-Meier method. In addition, multivariate analysis suggested PLK4 as an independent predictor of overall survival (HR, 0.556; 95%CI, 0.37620.822; P = 0.003) and disease-free survival (HR, 0.547; 95%CI, 0.38220.783; P = 0.001). Collectively, our study demonstrated that PLK4 was remarkably downregulated in HCC and could be served as a potential prognostic marker for patients with this deadly disease.
Funding: This work was supported by grants from the National Natural Science Foundation of China (No. 81172345 and No. 30973506). The funder 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.
Hepatocellular carcinoma (HCC) is the fifth most prevalent
cancer, thirdly leading cancer-related death worldwide . The
mortality rate of HCC has been increasing in China since the
1990 s, and HCC has became the second leading cause of cancer
death . To date, many risk factors, such as hepatitis B or C viral
infection, alcohol consumption, aatoxinB1, and genetic
predisposition have been identified as causes of HCC [3,4,5,6]. However,
the pathogenic mechanism and inadequacy of early detection of
HCC have not been clearly clarified. On the other hand, high
incidence of recurrence and metastasis are the primary reasons of
poor prognosis of HCC . As a result, a large series of
investigations are focused on the discovery of biological markers
useful for HCC diagnosis and prognostic prediction to provide
scientific guidance to clinical management.
Polo-like kinases (PLKs) play essential roles in cell cycle
progression [8,9,10,11,12], and DNA damage response [13,14].
Polo-like kinase 4 (PLK4), originally identified in Drosophila as a
serine/threonine kinase and mapped to chromosome 4 q28 which
is a region frequently associated with loss of heterozygosity (LOH)
in hepatoma , is essential for centriole duplication and cell
cycle progression [16,17]. PLK4 gradually increases from G1
phase and peaks in mitosis, suggesting that its expression is
regulated in a cell cycle dependent fashion [18,19]. In human
cancers, PLK4 is differently expressed. For example, PLK4 was
demonstrated to be downregulated in HCC [20,21], but
upregulated in colorectal cancer . Dysregulation of PLK4
caused disturbance of centrosome duplication, which may
ultimately resulted in occurrence of tumor . Findings that
PLK4 was reduced during hepatocarcinogenesis due to the
promoter hypermethylation , and that decrease of PLK4
contributed to HCC development in PLK4 mutant mice 
suggest reduced PLK4 expression may associate with HCC
carcinogenesis. Furthermore, PLK4 homozygous null mice were
embryonic lethal at E7.5 with a marked increase in mitotic and
apoptotic cells . Less chances of bearing spontaneous lung and
liver cancer were recorded in PLK4+/+ mice, compared with
PLK4+/2 littermates .
In this study, we examined PLK4 expression in HCC cell lines
and human tissues, analyzed the correlation between PLK4
expression and clinicopathological characteristics, and determined
the role of PLK4 in HCC prognostic prediction. Our data
indicated that PLK4 was remarkably decreased in HCC and could
be served as a promising biomarker of prognosis.
Materials and Methods
L02, MiHA and Huh7 cell lines were purchased from American
Type Culture Collection (ATCC, Manassas, VA). SMMC-7721,
Bel-7404, Bel-7402 and QSG-7703 cell lines were obtained from
the Type Culture Collection Cell Bank, Chinese Academy of
Science Committee (Shanghai, China). L02 was maintained in
RPMI 1640 with 15% of fetal bovine serum (FBS), 100 U/ml of
penicillin, and 100 U/ml of streptomycin. SMMC-7721,
Bel7404, Bel-7402 and QSG-7703 cells were cultured in RPMI 1640
with 10% of fetal bovine serum (FBS), 100 U/ml of penicillin, and
100 U/ml of streptomycin. MiHA and Huh7 cells were cultured
in Dulbecco modified Eagle medium (DMEM) containing 10% of
fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 of U/ml
streptomycin. All cell lines were incubated in a humidified
atmosphere of 5% CO2 and 95% air at 37uC.
Patients and tissue specimens. All HCC specimens along
with complete clinical and pathological data were obtained from
246 HCC patients who underwent surgical resection at Sun
YatSen University Cancer Center (SYSUCC), Guangzhou, China,
between Feb 1997 and Dec 2001. Twenty paired HCC and
corresponding adjacent nontumorous tissues immersed in
RNAlater (Ambion, Inc., USA) immediately after surgical resection and
stored at 280uC were subjected to quantitative real-time RT-PCR
and western blot. 219 males (91.3%) and 27 females (8.7%)
comprise of the 246 patients aged from 14 to 78 years (median age
is 48). None of the patients had received adjuvant therapies before
surgery. Tumor stage was defined according to
tumor-nodemetastasis (TNM) classification of the American Joint Committee
on International Union against Cancer. Tumor differentiation was
assessed according to Edmonson and Steiner grading system. The
use of tissues for this study has been approved by the Institute
Research Medical Ethics Committee of SYSUCC.
Tissue microarray (TMA) construction. According to the
method described previously , we constructed the tissue
microarray, containing of 246 HCC and adjacent nontumorous
tissues. In brief, all specimens were fixed in 10% formalin and
embedded in paraffin. The corresponding histological HE-stained
sections were reviewed by two pathologists to mark out
representative areas. Using a tissue arraying instrument (Beecher
Instruments, Sliver Spring, MD), each tissue core with a diameter
of 0.6 mm was punched from the marked areas and re-embedded.
RNA preparation and quantitative real-time PCR. Total
RNA was extracted from 20 pairs of HCC samples, following the
Trizol reagent (BIOO Scientific Co., USA) manufacturers
instruction. mRNA was reversed to cDNA by M-MLV Reverse
Transcriptase (Promega Inc., USA). The levels of PLK4 and
bactin were measured by SYBR green-based real-time PCR using
the Stratagene Mx3000P Real-Time PCR system. Primers were
designed as follows: PLK4, Forward:
AATCAAGCACTCTCCAATC and Reverse: TGTGTCCTTCTGCAAATC; b-actin,
Forward: TGGCACCCAGCACAATGAA and Reverse:
CTAAGTCATAGTCC GCCTAGAAGCA. Conditions were
set as follows: one cycle of 95uC for 10 min, followed by 40
amplification cycles at 95uC for 10 s, annealing at 60uC for 20 s
and elongation at 72uC for 15 s. Using the comparative threshold
cycle (22DDCt) method , the relative expression of PLK4 in
HCC were normalized to the endogenous b-actin.
Western blot. Total protein was extracted from 20 pairs of
HCC fresh tissues. 30 ug of protein was loaded onto 8%
SDSPAGE and transferred to PVDF membranes. After blocking, the
membranes were incubated with primary antibody against PLK4
(1:2000 dilutions, rabbit anti-PLK4, Epitomics Biotechnology,
Inc., USA). The membranes were then incubated with horseradish
peroxidase-linked mouse anti-rabbit antibody (at a 1:3000
dilution, Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.,
USA). GAPDH was served as a loading control.
Immunohistochemistry. Immunohistochemistry (IHC)
analysis for PLK4 was performed using a standard two-step
method . TMA sections were baked overnight at 37uC, and
then deparaffinized and rehydrated. Slides were boiled in Ethylene
Diamine Tetraacetic Acid (EDTA; 1 mmol/L; PH 8.0) in a
pressure cooker for antigen retrieval. Subsequently, slides were
incubated overnight at 4uC with PLK4 antibody (1:1600 dilution,
goat polyclonal antibody, Santa Cruz Biotechnology, Inc., Santa
Cruz, Calif., USA). After rinsed with PBS, the slides were
incubated with a secondary antibody and stained with 3,
3diaminobenzidine tetrahydrochloride (DAB). Finally, the slides
were counterstained with Mayers hematoxylin. Slides
immunoreacted with PBS were used as the negative controls.
Immunohistochemistry evaluation. Semi-quantitative
IHC detection was used to determine the PLK4 protein levels.
Using the H-score method , we multiplied the percentage
score by the staining intensity score. The percentage of
positivelystained cells was scored as 0 (0%), 1 (1%25%), 2 (26%
50%), 3 (51%75%), 4 (76%100%). Intensity was scored as
0 (negative staining), 1 (weak staining), 2 (moderate
bpatients were divided according to the median age; AFP, alpha-fetoprotein; HBsAg, hepatitis B surface antigen; PLK4, polo-like kinase 4.
staining), and 3 (strong staining).The median H-score was
chosen as cutoff point to separate high PLK4 expression
(Hscore.median) from low PLK4 expression (H-score # median)
Statistical analysis. Statistical analyses were performed
using the SPSS 16.0 software (SPSS, Chicago, IL, USA). The
Students t test was used for comparison between groups. The x2
test was performed to analyze the correlation between PLK4
expression and clinicopathological parameters. The Kaplan-Meier
method (the log-rank test) was used for survival curves. Cox
regression model with stepwise manner (forward, likelihood ratio)
was utilized to perform a multivariate analysis. P,0.05 (two-tailed)
was considered statistically significant.
The Expression of PLK4 in HCC Cell Lines
To examine the expression of PLK4 in HCC, we firstly detected
its mRNA level in immortalized liver cell lines and HCC cell lines.
According to the results of qRT-PCR, levels of PLK4 mRNA in
HCC cell lines were noticeably lower than those in immortalized
liver cells (Fig. 1A). We next determined the protein expression of
PLK4. As depicted in the result of western blot, PLK4 was
markedly downregulated in most of the tested HCC cells,
compared to that of L02 cells (Fig. 1B).
The mRNA and protein levels of PLK4 in HCC
tissues. We next examined PLK4 expression in 20 paired
HCC and the corresponding adjacent nontumor tissues, using
qRT-PCR and western blot. In 13 out of 20 cases, PLK4 mRNA
was downregulated in tumor tissues, compared with the adjacent
nontumorous tissues (Fig. 2A). PLK4 mRNA expression was
remarkably higher in 3 cases while remained unchanged in the rest
samples. Consistently, in 75% of cases, the protein levels of PLK4
in HCC tissues were dramatically lower than those in the
nontumorous tissues (Fig. 2C), as depicted in the result of western
blot, using the same HCC samples for qRT-PCR. The altered
expression of PLK4 between tumor and adjacent nontumor tissues
appeared statistically significant (Fig. 2B&D).
The relationship between PLK4 expression and
clinicopathological parameters. IHC was performed to
assess the expression of PLK4 in 246 paraffin-embedded HCC
tissues. Results revealed that PLK4 expression was mainly present
in the cytoplasm of cancer cells (Fig. 3AE) Scattered staining of
PLK4 in nuclear was also observed. As indicated by Figure 3F, low
PLK4 expression in tumor tissue was found in 175 out of 246
The relationship between PLK4 expression and
clinicopathological parameters was further analyzed. Significant correlations
were found between PLK4 expression and three parameters
including clinical stage (P = 0.034), serum AFP positive (P = 0.019)
and tumor size (P = 0.032). HCC patients with low PLK4
expression had a higher tendency to be with advanced stage,
high level of serum AFP and large-size tumor. There were no
statistical connections between PLK4 expression and the rest
clinicopathological parameters, such as age, gender, HBsAg,
cirrhosis, differentiation and vascular invasion (P.0.05, Table 1).
The association of low PLK4 expression in HCC with poor
survival. The association between PLK4 expression in HCC
and the survival of selected patients was analyzed with
KaplanMeier survival analysis. Patients with low PLK4 expression were
likely to be with significantly shorter overall survival (P = 0.002,
Fig. 4A), disease-free survival (P = 0.012, Fig. 4B) and
metastasisfree survival (P = 0.003, Fig. 4C), but not recurrence-free survival
(P = 0.121, Fig. 4D).
Since PLK4 expression was significantly corrected to AFP,
tumor size and clinical stage, we further determined the
relationship between PLK4 expression and the survival of patients
subclassified as large tumor, AFP ($20 ng/ml) and Stage (III
VI). As showed by Figure 5, patients in subclassified groups with
low PLK4 expression survived shorter than those with high PLK4
Univariate and multivariate analyses of prognostic
variables in HCC Patients. We next evaluated the expression
of PLK4 and other clinicopathologic parameters on prognosis of
HCC, using univariate analyses. Results indicated that PLK4, as
well as serum AFP level, tumor size, tumor multiplicity, tumor
differentiation, clinical stage and vascular invasion, was
responsible for efficacy of surgical treatment in HCC patient, by showing
that PLK4 expression was significantly associated with overall
survival (P = 0.002) and disease-free survival (P = 0.014) of HCC
patients (Table 2).
Furthermore, PLK4 expression and those clinicopathologic
variables significant in univariate analysis (i.e., serum AFP level,
tumor size, tumor multiplicity, tumor differentiation, clinical stage
and vascular invasion) were further evaluated in multivariate
analysis. Results suggested that PLK4 was also an independent
predictor for overall survival (HR: 0.556, 95% CI: 0.37620.822,
P = 0.003) and disease-free survival (HR: 0.547, 95% CI:
0.38220.783, P = 0.001) of HCC patients (Table 3).
Polo-like kinases (PLKs) are important regulators of cell cycle
progression, mitosis, cytokinesis, and DNA damage response
[8,9,10,11,12,14]. A previous study showed that both mRNA and
protein expressions of PLK4 gradually decreased from normal
liver to HCC . In this study, to elucidate the clinical role of
PLK4 in HCC, we applied TMA and IHC to examine its
expression in a cohort of Chinese patients. A significant decline of
PLK4 expression was observed in HCC tissues, compared with the
adjacent nontumorous tissues. To our knowledge, this is the first
study to analyze PLK4 expression in HCC using TMA-based IHC
Clinically, correlation of PLK4 expression with
clinicopathological parameters was also determined in previous studies.
Reduced expression of PLK4 was reported to connect with age
,0.001 2.595(1.80923.723) ,0.001
P value HR (95% CI)
AFP (ng/ml) 2.951(1.93324.506)
,0.001 2.725(1.86523.982) ,0.001
Liver cirrhosis 1.110(0.73121.685)
,0.001 2.142(1.49723.064) ,0.001
,0.001 1.854(1.47322.333) ,0.001
,0.001 1.612(1.30821.987) ,0.001
,0.001 3.991(2.59726.134) ,0.001
CI, confidence interval; HR, hazard ratio; AFP, alpha-fetoprotein; HBsAg,
hepatitis B surface antigen; PLK4,polo-like kinase4.
in colorectal tumor . Decreased expression of PLK4 in HCC
was associated with larger tumor size, indicating that the loss of
PLK4 may facilitate HCC growth. This adds to the previous
findings reporting the central role of PLK4 in mitotic regulation
 and centriole duplication . In the present study, we found
low expression of PLK4 in HCC was significantly associated with
other malignant tumor characteristics, such as advanced stage and
high serum AFP, indicating that PLK4 might be served as a
hallmark of advanced stage tumors. This could be supported by
the findings that constitutive expression of murine PLK4
suppressed cell growth , and that downregulation of PLK4
antagonized the antiproliferative and proapoptotic effects in
nontumor cells . Collectively, PLK4 may have antitumor
properties. Monitoring the expression dynamics of PLK4 may
contribute to characterize patients as frequently monitored ones or
adjuvant therapy given ones.
In results of Kaplan-Meier survival analysis, we found that
patients with high PLK4 expression had longer survival. This
might be explained by the findings that high PLK4 expression
positively correlated with a higher proliferation index , and
that high proliferative tumors could better response to therapy.
Furthermore, Cox regression analysis suggested PLK4 as an
independent prognostic factor, indicating that PLK4 may be a
pivotal modulator involved in cancer development. This could be
supported by other reports showing that decreased PLK4 led to
dysregulation of cell proliferation , and that PLK42/2
embryos were arrested at E7.5 and ultimately dead . On the
other hand, recent studies provided a comprehensive
understanding of PLK4s role in tumor progression and cell differentiation.
Elderly PLK4+/2 mice usually had more chances to develop
spontaneous liver and lung cancer . Furthermore, PLK4
controlled the differentiation of trophoblast stem cells into
trophoblast giant cells in vitro . Collectively, downregulation
Hazard ratio (95%CI) P value
b, Regression coefficient; SE, standard error; CI, confidence interval; AFP,
alphafetoprotein; PLK4, polo-like kinase4.
of PLK4 in tumor tissues may facilitate tumor progression,
subsequently leading to poor prognosis.
Stratified survival analysis of HCC, according to clinical stage,
tumor size and serum AFP, evaluated PLK4 expression to be
closely correlated with survival of HCC patients with stage IIIIV,
larger tumor size and high-level AFP. This suggested that
downregulation of PLK4 in HCC could be of clinical use for
distinguishing a set of patients with poor prognosis. Tumor
differentiation and vascular invasion and serum AFP are the most
commonly agreed survival indices for affecting the prognosis of
HCC patients [31,32,33]. But these three parameters have
different types of limits in offering critical information affecting
patient prognosis. In this study, our results indicated PLK4
expression evaluated by IHC could be used as an additional tool in
identifying those patients at risk of HCC progression.
Taken together, our study provides compelling clinical evidence
that PLK4 can be served as an independent prognostic marker for
overall survival and disease-free survival in HCC. Decreased
PLK4 expression in HCC is significantly related to tumor size,
serum AFP, and clinical stage. Although the current results which
are based on a cohort of Chinese patients should be further
confirmed in other HCC cohorts, our findings suggest PLK4 as a
new and promising prognostic biomarker for HCC progression
Conceived and designed the experiments: JPY CZYZ. Performed the
experiments: LLL CZYZ MYC JF. Analyzed the data: LLL CZYZ MYC
GGC. Contributed reagents/materials/analysis tools: MYC. Wrote the
paper: LLL CZYZ JPY.
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