Prognostic Value of Twist-1, E-cadherin and EZH2 in Prostate Cancer: An Immunohistochemical Study
Aziza E. ABDELRAHMAN1, Shimaa A. ARAFA1, Ragab A. AHMED2
1Department of Pathology, Zagazig University, Faculty of Medicine, ZAGAZIG, EGYPT
2Department of Urology, Zagazig University, Faculty of Medicine, ZAGAZIG, EGYPT
Keywords: EZH2, Twist-1, E-cadherin, Prostate cancer, Epithelial-mesenchymal transition
There is an urgent need to identify molecular biomarkers rather than clinical markers to distinguish aggressive prostate cancer from
the indolent majority for proper treatment and accurate prediction of prognosis. We aimed to investigate the immunohistochemical expression
of epithelial-mesenchymal transition (EMT)-related molecules (Twist-1 and E-cadherin) and the stem cell marker EZH2 in prostate cancer and
to assess their ability to identify the high-risk patients, in a trial to explore their prognostic implications.
Material and Method: Immunohistochemical expression of Twist-1, E-cadherin and EZH2 in 50 specimens of prostate cancer and 20 cases
of benign prostatic hyperplasia were studied. The relationship between their expression and the clinicopathological variables, biochemical
recurrence, and biochemical progression-free survival were investigated.
Results: Our results revealed that high Twist-1, as well as high EZH2 expression, was strongly associated with higher pre-treatment PSA level,
Gleason score ≥7, advanced tumor stage, lymph node involvement, distant metastasis and biochemical progression. Aberrant E-cadherin
expression was significantly associated with higher pre-treatment PSA level, Gleason score ≥7, advanced tumor stage, lymph node involvement,
and distant metastasis. A significant positive correlation between Twist-1 and EZH2 expression was found (p<0.001), while E-cadherin expression
showed a negative correlation with both markers (p<0.001). A significant association was found between high Twist-1, high EZH2& aberrant
E-cadherin expression, and shorter biochemical progression-free survival.
Conclusion: The high Twist-1 expression, aberrant E-cadherin and high EZH2 expression in primary prostate cancer are considered as adverse
prognostic markers of an aggressive tumor with high metastatic potential. Assessment of their expression level would contribute to the accurate
prediction of biochemical progression.
Prostate cancer (PC) is the most common neoplasm of
males and despite the progressive decline in its incidence
and mortality, it is still the second common cause of cancerrelated
death among men1
. The National Comprehensive
Cancer Network (NCCN) guidelines defined the high-risk
localized prostate cancer as initial PSA >20 ng/ml, clinical
stage ≥T3a and Gleason score ≥82
. However, these clinical
markers do not adequately discriminate between indolent
tumors and those that will progress to be metastatic, so
efforts are now directed towards using a combination of
biological rather than clinical markers to predict prognosis
and response to therapy3
Several studies have reported that epithelial-mesenchymal
transition (EMT) and cancer stem cells (CSCs) play a key
role in metastasis and drug resistance of prostatic carcinoma.
Understanding the determinants responsible for EMT and
CSCs will be essential to develop new promising therapies
for PC in the future4.
Epithelial-mesenchymal transition is a process found in
embryogenesis and tumor invasion which is thought to
be a general character of cancer stem cell and progenitor
cell populations. They lose their polarity and intercellular
adhesion molecules to transform into invasive and
migrating mesenchymal cells5.
Castration can induce EMT that may enhance the stemness
of CSCs to facilitate aggressive and metastatic behavior,
which in turn results in castration-resistance and metastasis6. Recent studies have demonstrated that EMT plays a
critical role in tumor recurrence and that it is tightly linked
to the biology of cancer stem cells or cancer-initiating cells7.
The major changes of epithelial-mesenchymal transition
occur at the molecular level before tumor morphology
and the utilization of these early molecular changes may
be crucial in predicting the prognosis of cancer8. The
process of EMT includes upregulation of pro-EMT proteins
such as Twist-1, N-cadherin and fibronectin, as well as down-regulation of EMT depressors as desmoplakin,
cytokeratins, and E-cadherin9.
Twist-1, a member of the basic helix-loop-helix transcription
factors, was reported as a key factor in EMT promoting
metastasis of a cancer cell. Previous studies have been
reported that Twist-1 is a master regulator of embryonic
morphogenesis that controls cell migration and differentiation
under various physiological conditions and promotes
EMT under some pathological conditions including cancer10.
The Twist-1 biomarker plays a crucial role in tumor growth,
cell invasion, and metastasis through regulation of cancerrelated
functions, such as angiogenesis, and degradation
of the extracellular matrix in various malignancies. In
addition, it promotes EMT by repressing the expression of
E-cadherin leading to disassembly of adherens junctions
and increased migratory potential11.
E-cadherin is a Ca˛-dependent transmembrane protein
that mediates cellular adhesion in normal epithelium via
interactions with β-catenin in the cytoplasm. Its expression
level is negatively correlated with the development of
EMT and tumor invasion. Loss or aberrant expression of
E-cadherin is related to PC progression, metastasis, and
poor prognosis through two different mechanisms, cell-cell
adhesion and paracrine action12. Cleavage of E-cadherin
ectodomain has been shown to create an sE-cad fragment,
capable of inducing EMT, invasion, and proliferation in a
paracrine manner via EGFR signaling13.
Epithelial-mesenchymal transition could potentially offer
a satisfactory explanation for the origin of CSCs, but the
molecular mechanisms linking EMT to stemness are still
unclear. The cancer stem cell theory contributes to a second
explanation for the relapse and resistance that occurs in
multiple tumors after therapy7.
Enhancer of zeste homolog-2 (EZH2), a catalytic subunit
of Polycomb Repressive Complex 2 (PRC2) responsible
for histone H3-lysine 27 methylation, regulates gene
transcription and chromatin structure. Some studies suggest
that EZH2 plays a crucial role in stem cell renewal, maintenance,
and differentiation into specific cell lineages14.
Moreover, it is overexpressed in aggressive solid tumors,
including prostate cancer, and may be a useful prognostic
marker in clinical practice with chemotherapeutic agents
that specifically target the enzyme15. Some studies have
also suggested that its oncogenic activity is thought to be
mainly mediated by silencing tumor suppressor genes. EZH2 is implicated in EMT activation by the inhibition of
E-cadherin expression, and its upregulation represents one
of the most frequent epigenetic alterations during prostate
Development of drugs either inhibiting EMT, CSCs or
enhancing the expression of epithelial markers could be a
novel strategy for PC therapy in the future4. In the present
study, we aimed to investigate the immunohistochemical
expression of EMT-related molecules (Twist-1 and
E-cadherin) and the stem cell marker EZH2 in PC and to
assess their ability to identify high-risk patients, in a trial to
explore their prognostic implications.
In this retrospective cohort study, fifty formalin-fixedparaffin-
embedded tissue specimens of PC and twenty
cases of benign prostatic hyperplasia (BPH) as control cases
were collected from the archive of Pathology Department,
Zagazig University Hospitals, Egypt. The tissue specimens
were obtained either by transrectal ultrasound-guided
prostatic needle biopsy (15 cases, 6 paraffin blocks for each),
open prostatectomy (5 cases), or radical prostatectomy (30
cases) at the Urology Department during the period from
November 2013 to December 2015. The clinicopathological
data were retrieved from patients’ files including age; initial
prostate-specific antigen (PSA) level, tumor stage (pT),
Gleason score, nodal metastasis, distant metastasis and
follow-up data. Distant metastasis was established by clinical
features, radiology, and radio-isotopic bone scan. Metastasis
to lymph nodes was diagnosed by histological examination
beside PSA staining of the resected lymph nodes. None of
the patients had received preoperative androgen ablation
before surgery. Histopathologic diagnosis of all cases
was reviewed by two pathologists (AE, SA) to unify the
reproducibility of the diagnosis. According to World
Health Organization classification of prostate cancer and
the revised Gleason grading system, PCa specimens were
divided into low-grade (<7) and high-grade cancers (≥7)
(17) Tumor stages (pT) were defined based on the TNM
prostate cancer staging system (18). The mean follow-up
period of cancer patients was 23.32 ± 9.18 months (range
3-36 m). All patients were followed up with a digital
rectal examination, imaging studies and serum PSA assay
every 3 months in the first year, semi-annually thereafter.
Biochemical progression was defined as persistent or rising
PSA level of >0.2 ng/ml in two consecutive blood samples
over a 2-month period.
Paraffin sections of 3-5 μm were stained using the
streptavidin-biotin-peroxidase technique. The tissue
sections were deparaffinized in xylene and rehydrated
through graded alcohol. Epitope retrieval by boiling in
citrate buffer (pH 6.0) for 20 min was done and then washed
in phosphate buffer saline (PBS). Endogenous peroxidase
activity was blocked by incubation of slides in 3% hydrogen
peroxide for 20 minutes. After washing with PBS, blocking
serum was applied for 10 min. At room temperature, the
tissue sections were incubated overnight with an anti-EZH2
antibody (1:100, BD Biosciences, CA); Rabbit polyclonal
anti-Twist antibody (1:50, ab50581; Abcam, Cambridge,
UK) and monoclonal anti-E-cadherin antibody (1:50;
Dako, Carpinteria, CA, UK). After rinsing in PBS, the
tissues were incubated with a biotin-conjugated secondary
antibody (Lab Vision Corporation, Fermont, USA) and
then incubated using the streptavidin-biotin system for 1
hour at room temperature. The sections were incubated
with diaminobenzidine (DAB) for 15 minutes then rinsed
with distilled water. Finally, the slides were counterstained
with Meyer’s hematoxylin, dehydrated and mounted.
Negative controls were made by substitution of primary
antibodies with a non-immune serum. Normal prostatic
glands, thyroid cancer, and breast ductal carcinoma were
used as positive controls for E-cadherin, Twist-1, and
At the start of this study we excluded prostate cancer cases
showing marked heterogeneous appearance. In addition,
during the assessment of immunohistochemical expression
of the markers we evaluated four representative tumor
regions (high Gleason, low Gleason, central and margin) by
both extension and intensity.
Evaluation of Twist-1 Immunostaining
The cytoplasmic staining intensity was scored as follows: 0,
negative; 1, weak; 2, medium; and 3, strong. The staining
extent was scored as follows: 0, 0%; 1, 1-25%; 2, 26-50%;
3, 51-75%; and 4, 76-100%. The final scores (0-7) were
calculated as the sum of the intensity score and extent score.
The staining scores of more than 3 were considered as highexpression19.
Evaluation of E-Cadherin Immunostaining
Membranous E-cadherin immunoreactivity was interpreted
as normally or aberrantly immunoreactive. Immunoreactivity
was classified as normal if the specimens
showed strong or moderate membranous staining and
weak or negative cytoplasmic staining in >70% of the cells. Patterns other than those were regarded as aberrant immunoreactivity20.
Evaluation of EZH2 Immunostaining
The nuclear staining for EZH2 in ten high power fields was
recorded and calculated for each case and the percentage
of positive cells was scored as follows: 0-33%, score 1; 34-
66%, score 2 and >67%, score 3. The staining intensity (0:
negative; 1: mild; 2: moderate; 3: strong) was recorded.
Finally, the staining index (SI) was obtained by multiplying
the two scores: SI (0- 9). A final score of ≥4 was considered
to be high21.
Continuous variables were expressed as the mean ± SD &
median (range), and categorical variables were expressed as
a number (percentage). Continuous variables were checked
for normality by Shapiro-Wilk test. Mann-Whitney U-test
was used to compare between two groups of non-normally
distributed variables. Percent of categorical variables
were compared using Pearson’s Chi-square test or Fisher’s
exact test when appropriate. The trend of change in the
distribution of relative frequencies of ordinal data was
compared using the chi-square test for trend. Biochemical
progression-free survival (BPFS) was calculated as the
time from the start of treatment to date of biochemical
progression or the most recent follow-up contact that
a patient was known as biochemical progression free.
Stratification of BPFS was done according to markers.
These time-to-event distributions were estimated using
the Kaplan-Meier plot method, and compared using the
two-sided exact log-rank test. All tests were two-sided. A
p-value <0.05 was considered significant. Statistical analysis
was done using SPSS 22.0 for Windows (SPSS Inc., Chicago,
IL, USA) and MedCalc windows (MedCalc Software bvba
13, Ostend, Belgium). The study was approved by our local
research ethics committee.
The mean age of cancer patients at the diagnosis was
62.94±8.76 years, while the mean age of BPH cases was at
58.40±11.98 years. No significant difference between the
two groups concerning age was present (p=0.138). Gleason
score ≥ 7 was the most common score in this cohort (56%).
Forty percent of PC patients had pT2 disease while 60%
had pT3-pT4. Distant metastasis was detected in 12 cases
(24%) and lymph node involvement was observed in 24
cases (48%). During the follow-up period, biochemical
progression was noted in 30% of the patients. Mean serum PSA level in cancer patients was 19.8± 14.02 ng/ml (range
4-50) and 2.03±1.33 ng/ml (range 0.4-5) in BPH. Other
clinicopathological features of PC and BPH cases are
presented in Tables I
Click Here to Zoom
|Table I: Clinicopathological features of 50 patients with prostatic carcinoma
The majority of PC cases (54%) showed high Twist-1
expression in the tumor cells while 100% of BPH specimens
showed low expression (p<0.001). Further investigation of
the correlation between Twist-1 immunoexpression and
clinicopathological features revealed that the increased
expression was associated with an initial PSA level (p<0.001).
In addition, Twist-1 immunoreactivity was up-regulated
with increased tumor grade with statistically significant
difference (p<0.001) (Figure 1A-C). Furthermore, a
significant difference in Twist-1 expression was observed
regarding the pathological tumor stage (p=<0.001), LN
involvement (p<0.001), and distant metastasis (p=0.003).
Click Here to Zoom
|Figure 1: A) Low-grade prostate cancer (Gleason score <7) shows
negative immunoreactivity of Twist-1 (IHC; x400).
B) High-grade prostate cancer (Gleason score ≥7) shows low
cytoplasmic immunoreactivity of Twist-1 (IHC; x400).
C) High-grade prostate cancer (Gleason score ≥7) shows high
cytoplasmic immunoreactivity of Twist-1 (IHC; x400).
All BPH specimens showed moderate to strong
membranous immunoreactivity of E-cadherin (preserved pattern) (Figure 2), while 62% cases of PC specimens had
aberrant E-cadherin immunoreactivity with a significant
difference between the two groups (p<0.001).
Low-grade PC of Gleason score <7 showed aberrant
E-cadherin immunoreactivity in only 27.3% of cases,
while the majority of high-grade PC (89.3%) had aberrant
expression (p<0.001) (Figure 3A-D). A significant
correlation was found between E-cadherin expression
patterns and initial PSA level (p<0.001). Aberrant
E-cadherin expression was more common in advanced
pathological stages than the early stages (p<0.001).
Furthermore, loss of membranous E-cadherin expression
was found in all cases associated with LN involvement and
distant metastasis in contrast to those cases with negative
LN and without distant metastasis (p<0.001, p=0.002,
Click Here to Zoom
|Figure 2: Benign prostatic hyperplasia shows strong and complete
membranous immunoreactivity of E-cadherin (preserved
expression), (IHC; x400).
Click Here to Zoom
|Figure 3: A) Low-grade prostate cancer (Gleason score <7) shows preserved immunoreactivity of E-cadherin (IHC; x400). B) High-grade
prostate cancer (Gleason score ≥7) shows preserved immunoreactivity of E-cadherin (IHC; x400). C) High-grade prostate cancer (Gleason
score ≥7) shows aberrant membranous immunoreactivity of E-cadherin (IHC; x400). D) High-grade prostate cancer (Gleason score ≥7)
shows completely negative immunoreactivity of E-cadherin; note the normal immunoreactivity in the benign glands (IHC; x400).
Analysis of both staining intensity and extension of nuclear
EZH2 revealed that all cases of BPH showed a low expression
level limited to the prostate epithelium and absent in the
surrounding stroma, while 66% of PC cases exhibited a high expression level in tumor cells with a significant difference
between the two groups (p<0.001).
Analysis of the correlation between nuclear EZH2 and
clinicopathological parameters revealed that patients
with high EZH2 expression had a higher initial PSA level
and Gleason scores (p<0.001, p=0.001, respectively) at
presentation (Figure 4A,B). Moreover, it was found that all
cases of advanced tumor stage (pT3, pT4), LN involvement
and distant metastasis revealed high nuclear EZH2
Click Here to Zoom
|Figure 4: A) Low-grade prostate cancer (Gleason score <7) shows low nuclear immunoreactivity of EZH2 (IHC; x400). B) High-grade
prostate cancer (Gleason score ≥7) shows high nuclear immunoreactivity of EZH2 (IHC; x400).
The correlation between Twist-1, E-cadherin and EZH2
expression and the clinicopathological parameters are
demonstrated in Table III.
Click Here to Zoom
|Table III: The association between Twist-1, E-cadherin and EZH2 expression and clinicopathological parameters
Association Between Twist-1, E-Cadherin and EZH2
The correlation analysis of our marker expression among
PC cases revealed a significant positive correlation between
Twist-1 and EZH2 expression (p<0.001), while E-cadherin
expression showed a negative correlation with both markers
(p<0.001) (Table III).
Association of Twist-1, E-Cadherin and EZH2 Expression
with Biochemical Progression and BPFS
During the follow-up period, 15 out of 50 patients (30%)
showed biochemical progression at the mean time of
29.4 months with 3 year BPFS in 63.7% of cases. We
found that 51.9% of high Twist-1 expression, 38.7% of
aberrant E-cadherin, and 42.4% of high EZH2 cases had
a biochemical progression within 3 years. However, only 4.3% of low Twist-1 expression (p<0.001), 15.8% of normal
E-cadherin expression (p=0.086), and 5.9% of low EZH2
expression cases (p=0.008) had a progressive disease
(Table IV). Kaplan-Meier survival curve analysis revealed
a significant association between high Twist-1 (p<0.001),
aberrant E-cadherin (p=0.013) and high EZH2 expressions
(p=0.002) and shorter BPFS (Figure 5A-C).
Click Here to Zoom
|Table IV: Impact of markers on biochemical progression and biochemical progression free survival.
Click Here to Zoom
|Figure 5: Kaplan-Meier plots of biochemical progression free
survival (BPFS); A) Stratified according to Twist-1 expression
(p<0.001). B) Stratified according to E-cadherin expression
(p=0.013). C) Stratified according to EZH2 (p=0.002).
Prostate cancer is the second leading cause of cancerrelated
mortality in males. The transition of a subset of
tumors from indolent to invasive disease is associated
with a poor clinical outcome. Activation of EMT genetic
program is a major risk factor for cancer progression16
It is important to accurately predict the prognosis of cancer
patients following surgical resection of the tumors using
an accepted consensus to define postoperative follow-up
schedule as well as additional treatment strategies22
Therefore, individual assessment of a tumor’s aggressive
potential is crucial for clinical decision making. The critical
question is how to identify patients with a high-risk of
recurrence or progression23
In this study, we evaluated the expression level of Twist-1,
E-cadherin and EZH2 in PC in a trial to assess their role in
the pathogenesis, progression, and identification of cancer
patients who will likely progress to aggressive disease and
therefore need radical treatment or earlier intervention.
In the present work, Twist-1 up-regulation was observed
only in PC as compared to BPH; denoting its role in
neoplastic transformation and progression of cancer.
Increased expression of Twist-1 was associated with
the initial PSA level and Gleason grade supporting the
previous reports about its negative impact on cancer
cell differentiation and histological progression24.
However, Whiteland et al. reported that Twist-1 expression
was indifferent between PC and BPH. In addition, they
reported indifferent Twist-1 expression between different
Gleason scores. They explained this discrepancy of results
by different patient demographics25.
Furthermore, a significant positive association between
Twist-1 expression and TNM staging including pT stage,
LN involvement, and distant metastases was demonstrated
where most of the advanced stages had high Twist-1
expression. Our data agree with previous studies that found high Twist-1 expression in PC and its correlation with
disease aggressiveness and metastasis20,22.
The explanation for the significant association between
Twist-1 with advanced tumor stage and the metastatic
potential of the primary cancer is the transcriptional
repression of E-cadherin, resulting in a loss of cellular
adhesion and an enhancement of PC cell motility26.
These findings were confirmed by our results, where the
highest Twist-1 expression was significantly associated with
aberrant E-cadherin expression. In a previous in vitro study,
Kwok et al. reported that by reducing the expression of
Twist-1 in PC cell lines, E-cadherin was redistributed from
the cytoplasm to the cell membrane27. Consideration
of all these data, we can guess that Twist-1 overexpression in the PC may enhance EMT by promoting E-cadherin
Kwok et al. observed that down-regulation of Twist-1 in
androgen-independent PC cells increased their sensitivity
to anticancer therapy and decreased their invasion and
migration abilities, suggesting Twist-1 inactivation as a
potential strategy to control the growth and metastasis of
these cells27. Previous studies have reported that the
highest expression of Twist-1 was associated with high
grade, invasion, metastases, and therefore unfavorable
prognosis in different cancers as thyroid28, and lung
carcinomas29. These results regarding Twist-1 may help
to find a new therapeutic target to inhibit the invasion,
progression, and metastases in PC.
Loss of intercellular adhesion molecules has been accepted
as an initial step of malignant transformation preceding
lymphovascular invasion (LVI) and distant metastasis.
The key role of E-cadherin in carcinogenesis has been
established, particularly in the prostate30.
In the current study, all BPH showed preserved E-cadherin
immunoreactivity while 62% PC cases designated
aberrant E-cadherin immunoreactivity with a significant
difference between the two groups. The membranous
immunoreactivity of E-cadherin was negatively correlated
with the initial PSA level and increased Gleason score.
These results suggest that abnormal localization of E-cadherin may occur during PC progression leading to
loss of epithelial differentiation through the loss of cell
polarity and adhesion. Furthermore, aberrant E-cadherin
was significantly associated with advanced tumor stage,
LN metastasis, and distant metastasis; therefore with poor
prognosis. These results are hand in hand with Whiteland
et al.25. Meng et al. also reported that PC metastasis was
inhibited in mice that received a high dose of zileuton,
5-lipoxygenase inhibitor, which restored normal expression
On the other hand, Ipekci et al.’s analysis failed to demonstrate
an association between E-cadherin expression and tumor stage, grade, and initial PSA level. They explained their
results with the fact that EMT is an early event in tumor
progression and assessment of primary tumor does not give
any insight into the metastatic potential of cells that require
having other alterations to survive in distant tissues3.
Induction of EMT by different stimuli could generate
stem-like cells with enhanced self-renewal and invasive
capacity with a high drug resistance, which is strongly
associated with metastasis and recurrence. However, the
molecular mechanisms responsible for these processes are
not completely understood7.
EZH2 increases the proliferation and invasiveness of PC
cells32. The expression of EZH2 promotes the histone
deacetylase (HDAC) activity and the enhanced HDAC
activity leads to removal of the acetyl group from the
histone H3K27 at the E-cadherin promoter region. This
helps the histone methyl-transferase activity of EZH2. Trimethylation
of histone H3 on lysine 27 leads to chromatin
compaction and transcription factors suppression from
binding and initiating transcription (33). Moreover,
histone deacetylase inhibitors can prevent EZH2-
mediated suppression of E-cadherin and attenuation of cell
invasion, suggesting a possible mechanism of therapeutic
treatments. In addition, a large body of evidence suggests
that pharmacological inhibition of the enzymatic activity
of the methyl-transferase EZH2 is an attractive therapeutic
strategy for castration-resistant prostate cancer34.
In the present study, all BPH showed low EZH2 expression
while 66% PC cases showed high EZH2 expression with
a significant difference between the two groups; these
results are similar to the observation of Matsika et al.21.
Moreover, a significant association was found between
EZH2 with an initial PSA level, Gleason score, and TNM
staging of the prostate, which confirm its crucial role in the
promotion of carcinogenesis and progression as previously
reported by Bryant et al. who found that EZH2 increases the
proliferation and invasiveness of PC cells32. A significant
association between EZH2 and the different pathological
parameters had also been reported by some investigators21. This result contrasts with those of Varambally et al.
who did not find a significant correlation between EZH2
level and these pathologic parameters35. Laitinen et al.
also reported a significant association between EZH2 and
Gleason score and the initial PSA level, whereas a nonsignificant
association with tumor stage (pT) was found36.
In our study, we found a significant inverse association
between EZH2 and E-cadherin expression where 90.9% of cases with high EZH2 expression designated aberrant
E-cadherin expression whereas 94.1% of cases with low
EZH2 expression showed normal E-cadherin staining.
These findings confirm the suggestion of EZH2- mediated
repression of E-cadherin and explain the positive
correlation of EZH2 with advanced tumor stage through
the loss of cellular adhesion and the enhancement of
cancer cell motility. In addition, a significant association
between EZH2 and Twist-1 expression was noted in our
study confirming the observation of some investigator
regarding the ability of EMT (upregulation of Twist-1) to
generate stem like-cells (EZH2) enhancing the metastasis
and recurrence of the involved tumor7.
Regarding the association between the prognostic
parameters and biochemical progression in our study, we
found a significant association between the conventional
parameters including the initial PSA level, Gleason score
≥7, and tumor TNM stage with biochemical progression
as previously reported by other investigators37-39. In
addition, early biochemical recurrence was more prevalent
in cases with high Twist-1, aberrant E-cadherin, and high
In the present study, Kaplan-Meier survival curve analysis
revealed a significant association between the initial PSA
level, Gleason score, and TNM stage of the studied cases with
BPFS as previously reported22. In addition, a significant
association was found between high Twist-1 expression and
shorter BPFS. Similarly, other investigators have reported
that twist-1 expression confers a worse prognosis22.
Moreover, aberrant E-cadherin expression of PC cases
revealed shorter BPFS and therefore poor prognosis. These
results are hand in hand with other investigators22,25.
Whiteland et al. demonstrated that reduced E-Cadherin
expression was linked to worse prognosis and poor diseasefree
survival, where E-cadherin was the only EMT marker
significantly associated with patients who died as a result
of PC progression when its expression was lost25. On
the other hand, Ipekci et al. failed to find an association
between aberrant E-Cadherin expression and BPFS3.
As regards EZH2, cases with high expression revealed
shorter BPFS and so poor prognosis (p=0. 002). This finding
agreed with van Leenders et al. who confirmed that EZH2
expression in neoplastic cells had a high predictive value for
cancer outcome and had an increased risk of biochemical
progression (40). On the other hand, some have found
that EZH2 is not an independent prognosticator and when
combined with decreased expression of E-cadherin revealed
a significant association with short progression-free
survival41. These studies have used tissue microarrays (TMAs) and core biopsies which, by their nature, limit
tumor sampling and do not take into account the tumor
heterogeneity and patchy staining expected of stem cell
markers. The differences between our results and this
study may also be due to using different antibody clones.
Therefore, these observations support the suggestion that
EZH2 can serve as a beneficial biomarker for prognostic
purposes in a PC, as respect to risk evaluation and prognosis
prediction. EZH2 has also been suggested as a target for
PC immunotherapy as EZH2-based peptides are detected
by cytotoxic T cells and immunoglobulin G in PC patients42. Therefore, understanding the complex steps of EMT
and metastasis will help in the development of improved
anti-metastatic drug strategies that are helpful against the
circulating metastatic cells and therapy-resistant cancer
In conclusion, the high Twist-1 expression, aberrant
E-cadherin expression and high EZH2 in primary PC are
considered as adverse prognostic markers of an aggressive
tumor with high metastatic potential. Assessment of their
expression level would help in the accurate prediction of
the biochemical progression. Therefore, a standardized
clinical trial with a larger sample is required to assess the
value of these biomarkers as targeted therapy.
CONFLICT of INTEREST
The authors declare that they have no conflict of interest.
1) Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer
J Clin. 2016;66:7-30.
2) NCCN. Clinical practice guidelines in oncology: Prostate
cancer. National comprehensive cancer network 2014; Version 1:
Available from: NCCN.org. Detailed guidelines on the detection,
prevention, risk stratification and treatment of patients.
3) Ipekci T, Ozden F, Unal B, Saygin C, Uzunaslan D, Ates E.
Epithelial-mesenchymal transition markers β-catenin, snail,
and e-cadherin do not predict disease free survival in prostate
adenocarcinoma: A prospective study. Oncol Res. 2015;21:1209-16.
4) Li P, Yang R, Gao W. Contributions of epithelial-mesenchymal
transition and cancer stem cells to the development of castration
resistance of prostate cancer. Mol Cancer. 2014;13:55.
5) Celiŕ-Terrassa T, Meca-Cortés O, Mateo F, de Paz AM, Rubio N,
Arnal-Estapé A, Ell BJ, Bermudo R, Díaz A, Guerra-Rebollo M,
Lozano JJ, Estarás C, Ulloa C, Álvarez-Simón D, Milŕ J, Vilella
R, Paciucci R, Martínez-Balbás M, de Herreros AG , Gomis
RR, Kang Y, Blanco J, Fernández PL, Thomson TM. Epithelial
mesenchymal transition can suppress major attributes of human
epithelial tumor-initiating cells. J Clin Invest. 2012;122:1849-68.
6) Sun Y, Wang BE, Leong KG, Yue P, Li L, Jhunjhunwala S, Chen D,
Seo K, Modrusan Z, Gao WQ, Settleman J, Johnson L. Androgen
deprivation causes epithelial-mesenchymal transition in the
prostate: Implications for androgen-deprivation therapy. Cancer
7) Kong D, Li Y, Wang Z, Sarkar FH. Cancer stem cells and epithelialto-
mesenchymal transition (EMT)-phenotypic cells: Are they
cousins or twins? Cancers. 2011;3:716-29.
8) Lemma S, Karihtala P, Haapasaari KM, Jantunen E, Soini Y,
Bloigu R, Pasanen AK, Turpeenniemi-Hujanen T, Kuittinen
O. Biological roles and prognostic values of the epithelialmesenchymal
transition-mediating transcription factors Twist,
ZEB1and Slug in diffuse large B-cell lymphoma. Histopathology.
9) Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial
mesenchymal transition. Nat Rev Mol Cell Biol. 2014;15:178-96.
10) Qin Q, Xu Y, He T, Qin C, Xu J. Normal and disease-related
biological functions of Twist1 and underlying molecular
mechanisms. Cell Res. 2012;22:90-106.
11) Gajula RP, Chettiar ST, Williams RD, Thiyagarajan S, Kato Y, Aziz
K, Wang R, Gandhi N, Wild AT , Vesuna F, Ma J, Salih T, Cades
J, Fertig E, Biswal S, Burns TF, Chung CH, Rudin CM, Herman
JM, Hales RK, Raman V, An SS, Tran PTet. The twist box domain
is required for Twist1-induced prostate cancer metastasis. Mol
Cancer Res. 2013;11:1387-400.
12) Chunthapong J, Seftor EA, Khalkhali-Ellis Z. Seftor REB,
Amir S, Lubaroff DM, Heidger PM, Hendrix MJC. Dual roles
of E-cadherin in prostate cancer invasion. J Cell Biochem.
13) David J, Rajasekaran A. Dishonorable discharge: The oncogenic
roles of cleaved E-cadherin fragments. Cancer Res. 2012;72:2917-23.
14) Chou RH, Yu YL, Hung MC. The roles of EZH2 in cell lineage
commitment. Am J Transl Res. 2011;3:243-50.
15) Yang YA , Yu J. EZH2, an epigenetic driver of prostate cancer.
Protein Cell. 2013;4:331-41.
16) Nolan KD, Franco OE, Hance MW, Hayward SW, Isaacs JS.
Tumor-secreted Hsp90 subverts polycomb function to drive
prostate tumor growth and invasion. J Biol Chem. 2015;290:8271-82.
17) Epstein JI1, Allsbrook WC Jr, Amin MB, Egevad LL; ISUP
Grading Committee. The 2005 International Society of Urological
Pathology (ISUP) consensus conference on Gleason grading of
prostatic carcinoma. Am J Surg Pathol. 2005;29:1228-42.
18) Edge SB and Compton CC. The American Joint Committee on
Cancer: The 7th edition of the AJCC cancer staging manual and
the future of TNM. Ann Surg Oncol. 2010;17:1471-4.
19) Lei P, Ding D, Xie J, Wang L, Liao Q, Hu Y. Expression profile of
Twist, vascular endothelial growth factor and CD34 in patients
with different phases of osteosarcoma. Oncol Lett. 2015;10:417-21.
20) Yuen HF, Chua CW, Chan YP, Wong YC, Wang X, Chan KW.
Significance of TWIST and E-cadherin expression in the
metastatic progression of prostatic cancer. Histopathology.
21) Matsika A, Srinivasan B, Day C, Mader SA, Kiernan DM,
Broomfield A, Fu J, Hooper JD, Kench JG, Samaratunga H. Cancer
stem cell markers in prostate cancer: An immunohistochemical
study of ALDH1, SOX2 and EZH2. Pathology. 2015;47:622-8.
22) Behnsawy HM, Miyake H, Harada K, Fujisawa M. Expression
patterns of epithelial-mesenchymal transition markers in
localized prostate cancer: significance in clinicopathological
outcomes following radical prostatectomy. BJU Int. 2013; 111:30-7.
23) Hoogland AM, Kweldam CF, van Leenders LH. Prognostic
histopathological and molecular markers on prostate cancer
needle-biopsies: A review. Biomed Res Int. 2014;2014:341324.
24) Shiota M, Izumi H, Onitsuka T, Miyamoto N, Kashiwagi E,
Kidani A, Yokomizo A, Naito S, Kohno K. Twist promotes tumor
cell growth through YB-1 expression. Cancer Res. 2008;68:98-105.
25) Whiteland H, Spencer-Harty S, Thomas DH, Davies C, Morgan
C, Kynaston H, Bose P, Fenn N, Lewis PD, Bodger O, Jenkins
S, Doak SH. Putative prognostic epithelial-to-mesenchymal
transition biomarkers for aggressive prostate cancer. Exp Mol
26) Lee TK, Poon RT, Yuen AP Ling MT, Kwok WK, Wang XH, Wong
YC, Guan XY, Man K, Chau KL, Fan ST. Twist overexpression
correlates with hepatocellular carcinoma metastasis through
induction of epithelial-mesenchymal transition. Clin Cancer Res.
27) Kwok WK, Ling MT, Lee TW, Lau TC, Zhou C, Zhang X, Chua
CW, Chan KW, Chan FL, Glackin C, Wong YC, Wang X: Upregulation
of TWIST in prostate cancer and its implication as a
therapeutic target. Cancer Res. 2005; 65:5153-62.
28) Buehler D, Hardin H, Shan W, Montemayor-Garcia C, Rush PS,
Asioli S, Chen H, Lloyd RV. Expression of epithelial-mesenchymal
transition regulators SNAI2 and TWIST1 in thyroid carcinomas.
Mod Pathol. 2013; 26:54-61.
29) Zeng J, Zhan P, Wu G, Yang W, Liang W, Lv T, Song Y. Prognostic
value of twist in lung cancer: Systematic review and meta-analysis.
Transl Lung Cancer Res. 2015;4:236-41.
30) Lazari P, Poulias H, Gakiopoulou H, Thomopoulou GH, Barbatis
C, Lazaris AC. Differential immunohistochemical expression
of CD44s, E-cadherin and β-catenin among hyperplastic and
neoplastic lesions of the prostate gland. Urol Int. 2013;90:109-16.
31) Meng Z, Cao R, Yang Z, Liu T, Wang Y, Wang X. Inhibitor of
5-lipoxygenase, zileuton, suppresses prostate cancer metastasis by
upregulating E-cadherin and paxillin. Urology. 2013;82:1452.e7-14.
32) Bryant RJ, Cross NA, Eaton CL, Hamdy FC, Cunliffe VT. EZH2
promotes proliferation and invasiveness of prostate cancer cells.
33) Cao Q, Yu J, Dhanasekaran SM, Kim JH, Mani RS, Tomlins
SA, Mehra R, Laxman B, Cao X, Yu J, Kleer CG, Varambally S,
Chinnaiyan AM. Repression of E-cadherin by the polycomb
group protein EZH2 in cancer. Oncogene. 2008;27:7274-84.
34) Xu K, Wu ZJ, Groner AC, He HH, Cai C, Lis RT, Wu X, Stack EC,
Loda M, Liu T, Xu H, Cato L, Thornton JE, Gregory RI, Morrissey
C, Vessella RL,Montironi R, Magi-Galluzzi C, Kantoff PW, Balk
SP, Liu XS, Brown M. EZH2 oncogenic activity in castrationresistant
prostate cancer cells is Polycomb-independent. Science.
35) Varambally S, Dhanasekaran SM, Zhou M, Barrette TR, Kumar-
Sinha C, Sanda MG, Ghosh D, Pienta KJ, Sewalt RG, Otte AP,
Rubin MA, Chinnaiyan AM. The polycomb group protein
EZH2 is involved in progression of prostate cancer. Nature.
36) Laitinen S, Martikainen PM, Tolonen T, Isola J, Tammela TL,
Visakorpi T. EZH2, Ki-67 and MCM7 are prognostic markers in
prostatectomy treated patients. Int J Cancer. 2008;122:595-602.
37) Uemura H, K. Hoshino, T. Sasaki Miyoshi Y, Ishiguro H, Inayama
Y, Kubota Y. Usefulness of the 2005 International Society of
Urologic Pathology Gleason grading system in prostate biopsy
and radical prostatectomy specimens. BJU Int. 2009;103:1190-4.
38) Nonomura N, Takayama H, Nakayama M, Nakai Y, Kawashima
A, Mukai M, Nagahara A, Aozasa K, Tsujimura A. Infiltration of
tumour-associated macrophages in prostate biopsy specimens
is predictive of disease progression after hormonal therapy for
prostate cancer. BJU Int. 2011;107:1918-22.
39) Pierorazio PM, Ross AE, Lin BM, Epstein JI, Han M, Walsh
PC, Partin AW, Pavlovich CP, Schaeffer EM. Preoperative
characteristics of high-Gleason disease predictive of favourable
pathological and clinical outcomes at radical prostatectomy. BJU
40) van Leenders GJ1, Dukers D, Hessels D, van den Kieboom SW,
Hulsbergen CA, Witjes JA, Otte AP, Meijer CJ, Raaphorst FM.
Polycomb-group oncogenes EZH2, BMI1, and RING1 are
overexpressed in prostate cancer with adverse pathologic and
clinical features. Eur Urol. 2007;52:455-63.
41) Rhodes DR, Sanda MG, Otte AP, Chinnaiyan AM, Rubin MA.
Multiplex biomarker approach for determining risk of prostatespecific
antigen-defined recurrence of prostate cancer. J Natl
Cancer Inst. 2003;95:661-8.
42) Ogata R, Matsueda S, Yao A, NoguchiM, Itoh K, Harada M.
Identification of polycomb group protein enhancer of zeste
homolog 2 (EZH2)-derived peptides immunogenic in HLA-A24+
prostate cancer patients. Prostate. 2004;60:273-81.
43) Heerboth S, Housman G, Leary M, Longacre M, Byler S, Lapinska
K, Willbanks A, Sarkar S. EMT and tumor metastasis. Clin Transl