Introduction
Bladder cancer is the second most common urologic
malignancy amongst males (1). In
2015 in the United States, it was estimated that there were 74,000
new cases of bladder cancer and 16,000 deaths (2). Transitional cell carcinoma (TCC) or
urothelial carcinoma of the bladder (UCB) account for 90% of all
bladder cancer cases, and are divided in 2 variants: non-muscle
invasive or muscle invasive. The non-muscle invasive form has a
high rate of recurrence (50%), and it progresses to muscle invasive
form in ~11% of the cases (3).
Despite advances in multi-treatment approaches, including surgery,
radiotherapy and chemotherapy, the clinical outcomes of bladder
cancer patients remain unsatisfactory (4,5).
This is due to considerable tumor variability and heterogeneity,
even within the same pathological stage. Predicting a prognosis by
examining the clinicopathological characteristics remains
difficult. Therefore, identification of novel prognostic factors
with high sensitivity and specificity is important to evaluate the
prognosis of UCB and individualize treatment strategies.
CD147 is a member of the immunoglobulin superfamily.
It is widely expressed in human tumors and plays a central role in
the progression of many cancers by stimulating the secretion of
matrix metalloproteinases (MMPs) and cytokines (6). CD147 regulates cell proliferation,
apoptosis, metastasis, differentiation and chemical drug resistance
(6). In addition, recent studies
have demonstrated that the level of CD147 is associated with the
prognosis of patients in many cancers (6). Furthermore, CD147 is recognized as an
effective therapeutic target for hepatocellular carcinoma (HCC)
(7,8) and additional cancers (9,10),
and exciting clinical progress has been made in HCC treatment with
CD147-directed monoclonal antibodies (11).
The activator protein-1 transcription factor, AP-1,
is principally composed of homodimers of the Jun family (c-Jun,
JunB and JunD), heterodimers of the members of Fos (c-Fos, FosB,
Fra-1 and Fra-2) and Jun, or cAMP response element-binding
protein/activating transcription factor family members (12). Homo- or hetero- forms of the AP-1
complex can activate target gene expression by binding to
12-O-Tetradecanoylphorbol-13-acetate-(or TPA)-responsive elements,
which are also known as AP-1 sites (consensus sequence,
5-TGAG/CTCA-3), in promoter or enhancer regions (12). Evidence has demonstrated that AP-1
serves an important role in inflammation, cellular migration,
metastasis and cell proliferation, apoptosis and transformation
(13). More importantly, several
studies have revealed that the AP-1 complex is involved in
carcinogenesis of various tumor types, such as ovarian cancer,
breast cancer and skin cancer. For example, c-Fos has been
identified as independent predictor of decreased survival in breast
cancer (14). High levels of c-Fos
expression are associated with high-grade lesions and poor
prognosis in osteosarcoma and endometrial carcinoma (15,16).
High levels of c-Jun protein have been identified in 31% of primary
and metastatic lung tumors (17).
In breast cancer, activated c-Jun is expressed predominantly at the
invasive front of breast cancer and is associated with
proliferation and angiogenesis (18). However, there has been little
research into the levels of c-Jun and c-Fos in patients with
urothelial carcinoma of bladder tissues.
In the present study, the prognostic significance of
c-Fos, c-Jun and CD147 expression was examined by performing
immunohistochemistry with a tissue microarray of samples from 41
patients with UCB. In addition, the correlation between c-Fos,
c-Jun and CD147 expression in UCB was investigated to supply more
reliable judgments of the prognosis of UCB patients.
Materials and methods
Patients and tissues
A total of 41 bladder cancer tissues with
histologically confirmed UCB were obtained from the Shanghai Outdo
Biotech Co., Ltd. (Shanghai, China) from May 2007 to November 2011,
and 34 para-cancer normal tissues, which were 5 cm far away from
the edge of the tumor, were randomly selected as controls. The
study was approved by the Ethics Committee of the Fourth Military
Medical University (Xi'an, China). Following transurethral
resection and partial or radical cystectomy therapy, all patients
were available for follow-up data and provided written and informed
consent. Clinical pathological information was obtained from
patients' operative and pathological reports, and included: Sex,
age, recurrence, metastasis, invasion tumor size and growth
pattern, WHO grade, TNM stage, AJCC stage and survival time.
The study population comprised 35 men (85.4%) and 6
women (14.6%), with a median age at diagnosis of 71 years (range,
44–85 years). The tumor growth type was papillary in 11 patients
(26.8%) and nonpapillary in 30 patients (73.2%); low-grade tumors
were observed in 3 patients (7.3%) and high-grade tumors were
observed in 38 patients (92.7%). Furthermore, 6 patients (14.6%)
had low-stage (Tis) tumors and 35 patients (85.4%) had high-stage
tumors (T1, 6; T2, 10; T3, 15; T4a, 4; Table I). There was no recurrence or
distant metastasis recorded in all cases. Among these 41 patients,
18 (43.9%) did not survive. The median follow-up period was 35
months (data not shown).
 | Table I.CD147, c-Fos, and c-Jun expression in
41 patients with urothelial carcinoma of the bladder. |
Table I.
CD147, c-Fos, and c-Jun expression in
41 patients with urothelial carcinoma of the bladder.
|
| CD147 (%) |
|---|
|
|
|
|---|
| Factor | +++(n=3) (%) | ++(n=6) (%) | +(n=13) (%) | -(n=19) (%) | P-value |
|---|
| Age (y) |
| >71
(n=20) | 1 (5.0) | 2 (10.0) | 7 (35.0) | 10 (50.0) | 0.393 |
| ≤71
(n=21) | 2 (9.5) | 4 (19.0) | 6 (28.6) | 9 (42.9) |
| Sex |
|
|
|
|
|
| Male
(n=35) | 3 (8.6) | 6 (17.1) | 11 (31.4) | 15 (42.9) | 0.165 |
| Female
(n=6) | 0 (0) | 0 (0) | 2 (33.3) | 4 (66.7) |
| Growth pattern |
|
Papillary (n=11) | 0 (0) | 2 (18.2) | 5 (45.5) | 4 (36.4) | 0.964 |
|
Nonpapillary (n=30) | 3 (10.0) | 4 (13.3) | 8 (26.7) | 15 (50.0) |
| WHO grade |
|
|
|
|
|
| Low
(n=3) | 0 (0) | 1 (33.3) | 2 (66.7) | 0 (0) | 0.338 |
| High
(n=38) | 3 (7.9) | 5 (13.2) | 11 (28.9) | 19 (50.0) |
| Lymph node
invasion |
|
|
|
|
|
|
Negative (n=37) | 2 (5.4) | 6 (16.2) | 10 (27.0) | 19 (51.4) | 0.136 |
|
Positive (n=4) | 1 (25.0) | 0 (0) | 3 (75.0) | 0 (0) |
| TNM stage |
|
|
|
|
|
| Low
(Tis) (n=6) | 0 (0) | 0 (0) | 4 (66.7) | 2 (33.3) | 0.649 |
| High
(T1-T4a) (n=35) | 3 (8.6) | 6 (17.1) | 9 (25.7) | 17 (48.6) |
| Tumor diameter |
|
|
|
|
|
| >4
cm (n=16) | 0 (0) | 5 (31.3) | 6 (37.5) | 5 (31.3) | 0.355 |
| ≤4 cm
(n=25) | 3 (12.0) | 1 (4.0) | 7 (28.0) | 14 (56.0) |
| AJCC cancer
staging |
|
|
|
|
|
| Tis
(n=12) | 1 (8.3) | 1 (8.3) | 4 (33.3) | 6 (50.0) | 0.554 |
| 1–2
(n=11) | 0 (0) | 2 (18.2) | 4 (36.4) | 5 (45.5) |
| 3–4
(n=18) | 2 (11.1) | 5 (27.8) | 3 (16.7) | 8 (44.4) |
|
|
| c-Fos (%) |
|
|
|
| Factor | +++(n=0) | ++(n=1) (%) | +(n=9) (%) | −(n=31)(%) | P-value |
|
| Age (y) |
|
|
|
|
|
| >71
(n=20) | – | 0 (0) | 6 (30.0) | 14 (70.0) | 0.693 |
| ≤71
(n=21) | – | 1 (4.8) | 3 (14.3) | 17 (81.0) |
| Sex |
|
|
|
|
|
| Male
(n=35) | – | 1 (2.9) | 6 (17.1) | 28 (80.0) | 0.220 |
| Female
(n=6) | – | 0 (0) | 3 (50.0) | 3 (50.0) |
| Growth pattern |
|
|
|
|
|
|
Papillary (n=11) | – | 1 (9.1) | 1 (9.1) | 9 (81.8) | 0.973 |
|
Nonpapillary (n=30) | – | 0 (0) | 8 (26.7) | 22 (73.3) |
| WHO grade |
|
|
|
|
|
| Low
(n=3) | – | 0 (0) | 0 (0) | 3 (100.0) | 0.336 |
| High
(n=38) | – | 1 (2.6) | 9 (23.7) | 28 (73.7) |
| Lymph node
invasion |
|
|
|
|
|
|
Negative (n=37) | – | 1 (2.7) | 7 (18.9) | 29 (78.4) | 0.330 |
|
Positive (n=4) | – | 0 (0) | 2 (50.0) | 2 (50.0) |
| TNM stage |
|
|
|
|
|
| Low
(Tis) (n=6) | – | 1 (16.7) | 0 (0) | 5 (83.3) | 0.731 |
| High
(T1-T4a) (n=35) | – | 0 (0) | 9 (25.7) | 26 (74.3) |
| Tumor diameter |
|
|
|
|
|
| >4
cm (n=16) | – | 1 (6.3) | 3 (18.8) | 12 (75.0) | 0.651 |
| ≤4 cm
(n=25) | – | 0 (0) | 6 (24.0) | 19 (76.0) |
| AJCC cancer
staging |
|
|
|
|
|
| Tis
(n=12) | – | 1 (8.3) | 2 (16.7) | 9 (75.0) | 0.552 |
| 1–2
(n=11) | – | 0 (0) | 3 (27.3) | 8 (72.7) |
| 3–4
(n=18) | – | 0 (0) | 4 (22.2) | 14 (77.8) |
|
|
| c-Jun (%) |
|
|
|
| Factor | +++ (n=0) | ++ (n=0) | + (n=9) (%) | − (n=32) (%) | P-value |
|
| Age (y) |
|
|
|
|
|
| >71
(n=20) | – | – | 2 (10.0) | 18 (90.0) | 0.130 |
| ≤71
(n=21) | – | – | 7 (33.3) | 14 (66.7) |
| Sex |
|
|
|
|
|
| Male
(n=35) | – | – | 8 (22.9) | 27 (77.1) | 1.000 |
| Female
(n=6) | – | – | 1 (16.7) | 5 (83.3) |
| Growth pattern |
|
|
|
|
|
|
Papillary (n=11) | – | – | 2 (18.2) | 9 (81.8) | 1.000 |
|
Nonpapillary (n=30) | – | – | 7 (23.3) | 23 (76.7) |
| WHO grade |
|
|
|
|
|
| Low
(n=3) | – | – | 0 (0) | 3 (100) | 1.000 |
| High
(n=38) | – | – | 9 (23.7) | 29 (76.3) |
| Lymph node
invasion |
|
|
|
|
|
|
Negative (n=37) | – | – | 7 (18.9) | 30 (81.1) | 0.204 |
|
Positive (n=4) | – | – | 2 (50.00) | 2 (50.0) | – |
| TNM stage |
|
|
|
|
|
| Low
(Tis) (n=6) | – | – | 2 (33.3) | 4 (66.7) | 0.597 |
| High
(T1-T4a) (n=35) | – | – | 7 (20.0) | 28 (80.0) | – |
| Tumor diameter |
|
|
|
|
|
| >4
cm (n=16) | – | – | 5 (31.3) | 11 (68.8) | 0.276 |
| ≤4 cm
(n=25) | – | – | 4 (16.0) | 21 (84.0) | – |
| AJCC cancer
staging |
|
|
|
|
|
| Tis
(n=12) | – | – | 1 (8.3) | 11 (91.7) | 0.038 |
| 1–2
(n=11) | – | – | 1 (9.1) | 10 (90.9) | – |
| 3–4
(n=18) | – | – | 7 (38.9) | 11 (61.1) | – |
|
| – | – | – | – | – |
Immunohistochemical (IHC) staining and
evaluation
The expression of c-Jun, c-Fos and CD147 was
detected by IHC staining. Briefly, 5 µm sections were cut from
formalin-fixed, paraffin-embedded tissues. Following
deparaffinization and dehydration, sections were heated in an
autoclave (~120°C, 200 kPa, 2 to 6 min,) in 10 mM citrate buffer
(pH 6.0) for antigen retrieval, and treated with 3%
H2O2 for 20 min to block the peroxidase.
Sections were blocked in 5% goat serum in PBS, followed by
overnight incubation at 4°C with anti-c-Jun antibody (1:100
dilution; cat. no. #9165 Cell Signaling Technology, Inc., Danvers,
MA, USA), anti-c-Fos antibody (1:500 dilution; cat. no. #2251; Cell
Signaling Technology, Inc.) andanti-CD147 antibody (1:167 dilution;
cat. no. #201508; Cell Engineering Research Center, Fourth Military
Medical University, Xi'an, China). The signal was detected with
streptavidin-peroxidase staining kits (Thermo Fisher Scientific,
Inc., Waltham, MA, USA) and DAB substrate (ZSGB-BIO., Beijing,
China). All sections were then counterstained with Mayer's
hematoxylin (0.1%) for 5 to 8 min at room temperature (cat. no
#715012; Baso Diagnostics, Inc., Zhuhai, Guangdong, China) and then
mounted.
Images were taken with an inverted microscope
(CKX41; Olympus Corporation, Tokyo, Japan) equipped with a digital
camera under ×100 and ×400 magnification. Two pathologists
evaluated the results independently, with no knowledge of
clinicopathological features.
The expression of c-Jun, c-Fos and CD147 were scored
in a semi-quantitative manner according both the extent and the
intensity of the staining: IHC negative, absent; IHC +, faint or
weak intensity in >10% of cancer cells; IHC ++, moderate
intensity in >10% of cancer cells; and IHC +++, strong intensity
in >10% of cancer cells.
Statistical analysis
All statistical analyses were performed using SPSS
software (version, 17.0; SPSS Inc., Chicago, IL, USA). The
relationship between the level of three proteins (c-Fos, c-Jun and
CD147) and clinicopathological features was analyzed with
chi-squared statistical test (linear-by-linear association or the
Fisher exact test) or Spearman correlation analysis, while overall
survival (OS) analysis was carried out using the Kaplan-Meier
method and compared by the Log-rank test and the Cox proportional
hazards regression model served to identify relevant prognostic
factors. All P-values were two sided and P<0.05 was considered
to indicate a statistically significant difference.
Results
The expression characteristicss of
c-Jun, c-Fos and CD147 in UCB and normal tissues
A total of 9 of 41 (22.0%) UCB tissues and 1 of 34
(8.8%) para-cancer tissues presented positive staining of c-Jun,
respectively. Tumor staining of c-Jun expression was observed in
the nucleus staining pattern (Fig.
1), however, only negative or weak immunoreactivity of c-Jun
was detected in para-cancer tissues, which indicated that positive
c-Jun was more commonly expressed in human UCB than para-cancer
tissues (P=0.017; Fig. 1).
 | Figure 1.The immunohistochemical staining of
CD147, c-Fos and c-Jun in UCB. The expression of c-Jun, c-Fos and
CD147 was scored as negative, absence; +, weak; ++, moderate; and
+++, strong. The images were ×100 (upper row) and ×400
magnification (bottom row). The histogram presents the number of
cases with CD147, c-Fos and c-Jun expression in UCB tissues and
para-cancer normal tissues. The distribution of 41 cases of UCB and
34 cases of para-cancer tissues is presented in the bar graph.
CD147 (negative, 19; +, 3; ++, 6; and +++, 13 in UCB. negative, 34
in para-cancer), c-Fos (negative, 31; +, 9; and ++, 1 in UCB.
negative, 13; and +, 21 in para-cancer) and c-Jun (negative, 32; +,
9; in UCB. Negative, 33; and +, 1 in para-cancer), respectively.
***P<0.001, **P<0.01 and *P<0.05 vs. para-cancer. UCB,
urothelial carcinoma of the bladder. |
Following IHC in UCB tissues, 10 of 41 cases (24.4%)
presented c-Fos staining, which was strong in 1 case (2+ or higher,
2.4%), and weak in 9 cases (1+, 22.0%). The other tissues,
including 31 (75.6%) UCB and 13 of 34 (38.2%) normal bladder
tissues, demonstrated no staining. Tumor staining of c-Fos
expression was observed in the nucleus staining pattern (Fig. 1).
CD147 immunoreaction positive (+, ++, +++) was
observed in the 22 cases (53.7%) and most tumor cells with diffuse
plasma membranous staining of CD147, while none of normal
urothelial cells expressed CD147 (Fig.
1), which indicated that the CD147 was more common high
expression in human UCB than that in para-cancer normal tissues
(P<0.001; Fig. 1).
A weak inverse correlation was identified
(rho=−0.310, P=0.049; Table II)
existed in 41 UCB patients between c-Fos expression (negative, +,
++) and CD147 expression (negative, +, ++, +++). However, neither
c-Fos expression (rho =0.233, P=0.142) nor CD147 (rho =0.024,
P=0.881) expression had significant association with c-Jun
expression (negative, +).
| Table II.CD147, c-Jun and c-Fos expression
correlation in 41 patients with urothelial carcinoma of the
bladder. |
Table II.
CD147, c-Jun and c-Fos expression
correlation in 41 patients with urothelial carcinoma of the
bladder.
|
| CD147 | c-Jun | c-Fos |
|---|
|
|
|
|
|
|---|
| Marker | rho | P-value | rho | P-value | rho | P-value |
| CD147 | – | – | 0.024 | 0.881 | −0.310 | 0.049 |
| c-Jun | 0.024 | 0.881 | – | – | 0.233 | 0.142 |
| c-Fos | −0.310 | 0.049 | 0.233 | 0.142 | – | – |
Clinicopathological parameter
relevance and survival analysis
The protein expression of c-Jun was significantly
associated with AJCC cancer staging (P=0.038), while other
clinicopathological factors, including age, sex, growth pattern,
WHO grade, lymph nodes invasion, tumor node metastatis (TNM) stage
and tumor size, had no apparent relationship with c-Jun, c-Fos or
CD147 expression (Table I).
Neither CD147-c-Jun co-expression (5 cases in 41 UCB patients,
12.2%), CD147-c-Fos co-expression (2 in 41 UCB patients, 5.0%), nor
c-Fos-c-Jun co-expression (4 in 41 UCB patients, 9.8%) were
associated with any clinicopathological parameters (Table III).
| Table III.CD147, c-Fos, and c-Jun
co-expressions in 41 patients with urothelial carcinoma of the
bladder. |
Table III.
CD147, c-Fos, and c-Jun
co-expressions in 41 patients with urothelial carcinoma of the
bladder.
|
| CD147-c-Jun
co-expression |
|---|
|
|
|
|---|
| Factor | Co-expression (n=5)
(%) | Non-co-expression
(n=36) (%) | P-value |
|---|
| Age (y) |
|
|
|
| >71
(n=20) | 2 (10.0) | 18 (90.0) | 1.000 |
| ≤71
(n=21) | 3 (14.3) | 18 (85.7) |
| Sex |
|
|
|
| Male
(n=35) | 5 (14.3) | 30 (85.7) | 1.000 |
| Female
(n=6) | 0 (0) | 6 (100) |
| Growth pattern |
|
|
|
|
Papillary (n=11) | 1 (9.1) | 10 (90.9) | 1.000 |
|
Nonpapillary (n=30) | 4 (13.3) | 26 (86.7) |
| WHO grade |
|
|
|
| Low
(n=3) | 0 (0) | 5 (100) | 1.000 |
| High
(n=38) | 5 (13.2) | 33 (86.8) |
| Lymph nodes
invasion |
|
|
|
|
Negative (n=37) | 5 (13.5) | 32 (86.5) | 1.000 |
|
Positive (n=4) | 0 (0) | 4 (100) |
| TNM stage |
|
|
|
| Low
(Tis) (n=6) | 0 (0) | 6 (100) | 1.000 |
| High
(T1-T4a) (n=35) | 5 (14.3) | 30 (85.7) |
| Tumor diameter |
|
|
|
| >4
cm (n=16) | 0 (0) | 16 (100) | 0.137 |
| ≤4 cm
(n=25) | 5 (20.0) | 20 (80.0) |
| AJCC cancer
staging |
|
|
|
| Tis
(n=12) | 0 (0) | 12 (100) | 0.068 |
| 1–2
(n=11) | 1 (9.1) | 10 (90.9) |
| 3–4
(n=18) | 4 (22.2) | 14 (77.8) |
|
|
| CD147-c-Fos
co-expression |
|
|
|
| Factor | Co-expression (n=2)
(%) | Non-co-expression
(n=39) (%) | P-value |
|
| Age (y) |
|
|
|
| >71
(n=20) | 2 (10.0) | 18 (90.0) | 0.232 |
| ≤71
(n=21) | 0 (0) | 21 (100) |
| Sex |
|
|
|
| Male
(n=35) | 2 (5.7) | 33 (94.3) | 1.000 |
| Female
(n=6) | 0 (0) | 6 (100) |
| Growth pattern |
|
|
|
|
Papillary (n=11) | 2 (18.2) | 9 (81.8) | 0.067 |
|
Nonpapillary (n=30) | 0 (0) | 30 (100) |
| WHO grade |
|
|
|
| Low
(n=3) | 0 (0) | 3 (100) | 1.000 |
| High
(n=38) | 2 (5.3) | 36 (94.7) |
| Lymph nodes
invasion |
|
|
|
|
Negative (n=37) | 2 (5.4) | 35 (94.6) | 1.000 |
|
Positive (n=4) | 0 (0) | 4 (100) |
| TNM stage |
|
|
|
| Low
(Tis) (n=6) | 0 (0) | 6 (100) | 1.000 |
| High
(T1-T4a) (n=35) | 2 (5.7) | 33 (94.3) |
| Tumor diameter |
|
|
|
| >4
cm (n=16) | 1 (6.2) | 15 (93.8) | 1.000 |
| ≤4 cm
(n=25) | 1 (4.0) | 24 (96.0) |
| AJCC cancer
staging |
|
|
|
| Tis
(n=12) | 0 (0) | 12 (100) | 0.548 |
| 1–2
(n=11) | 1 (9.1) | 10 (90.9) |
| 3–4
(n=18) | 1 (5.6) | 17 (94.4) |
|
|
| c-Fos-c-Jun
co-expression |
|
|
|
| Factor | Co-expression (n=4)
(%) | Non-co-expression
(n=37) (%) | P-value |
|
| Age (y) |
|
|
|
| >71
(n=20) | 1 (5.0) | 19 (95.0) | 0.606 |
| ≤71
(n=21) | 3 (14.3) | 18 (85.7) |
| Sex |
|
|
|
| Male
(n=35) | 3 (8.6) | 32 (91.4) | 0.483 |
| Female
(n=6) | 1 (16.7) | 5 (83.3) |
| Growth pattern |
|
|
|
|
Papillary (n=11) | 0 (0) | 11 (100) | 0.559 |
|
Nonpapillary (n=30) | 4 (13.3) | 26 (86.7) |
| WHO grade |
|
|
|
| Low
(n=3) | 0 (0) | 3 (100) | 1.000 |
| High
(n=38) | 4 (10.5) | 34 (89.5) |
| Lymph nodes
invasion |
|
|
|
|
Negative (n=37) | 4 (10.8) | 33 (89.2) | 1.000 |
|
Positive (n=4) | 0 (0) | 4 (100) |
| TNM stage |
|
|
|
| Low
(Tis) (n=6) | 0 (0) | 6 (100) | 1.000 |
| High
(T1-T4a) (n=35) | 4 (11.4) | 31 (88.6) |
| Tumor diameter |
|
|
|
| >4
cm (n=16) | 0 (0) | 16 (100) | 0.143 |
| ≤4 cm
(n=25) | 4 (16.0) | 21 (84.0) |
| AJCC cancer
staging |
|
|
|
| Tis
(n=12) | 1 (8.3) | 11 (91.7) | 0.383 |
| 1–2
(n=11) | 0 (0) | 11 (100) |
| 3–4
(n=18) | 3 (16.7) | 15 (83.3) |
|
Univariate survival analyses revealed that high
CD147 expression (P=0.038), high c-Jun expression (P=0.008) and
high WHO grade (P<0.001; data not shown) were associated with
poor OS, whereas high c-Fos expression did not significantly
correlate with poor OS for UCB (P=0.225; Fig. 2A-C). Further analysis revealed that
either CD147-c-Fos-c-Jun co-expression (P=0.004) or CD147-c-Jun
co-expression (P=0.037) and c-Fos-c-Jun co-expression (P<0.001)
were also associated with poor OS (Fig. 2D-F). Therefore, it was considered
that OS was better in patients who had c-Fos, c-Jun and CD147 all
negative expression, or only one of them positive than in those who
had double positive or triple positive expression (P=0.008;
Fig. 2H).
 | Figure 2.OS analysis of c-Fos, c-Jun and CD147
expression in 41 patients with urothelial carcinoma of the bladder.
(A) Patients with CD147 positive expression (+, ++, +++) had poorer
overall survival rate than those with CD147 negative expression
(log-rank test, P=0.038). (B) Patients with c-Jun positive
expression (+) had poorer overall survival rate than those with
c-Jun negative expression (log-rank test, P=0.008). (C) c-Fos
positive expression (+, ++) did not significantly correlate with
poor OS (log-rank test, P=0.225). (D) Patients with all the three
proteins co-expressed (CD147-c-Fos-c-Jun co-expression) had poorer
overall survival rate than the others (log-rank test, P=0.004). (E)
Patients with CD147-c-Jun co-expression had poorer overall survival
rate than the others (log-rank test, P=0.037). (F) Patients with
c-Fos-c-Jun co-expression had poorer overall survival rate than the
others (log-rank test, P<0.001). (G) CD147-c-Fos co-expression
did not significantly correlate with poor OS (log-rank test,
P=0.768). (H) OS was better in patients who had c-Fos, c-Jun and
CD147 all negative, or single-protein positive expression than in
those who had double-protein positive or triple-protein positive
expression (log-rank test, P=0.008). OS, overall survival. |
Multivariate analyses (Table IV) demonstrated that high CD147
expression (hazard ratio [HR]=6.889; 95% confidence interval [CI]:
1.315–36.084; P=0.022), high c-Fos expression (HR=4.636; 95%
CI:1.128–19.057; P=0.033), high c-Jun (HR=4.589; 95%
CI:1.172–17.968; P=0.029) and even male sex (HR=0.140; 95%
CI:0.027–0.732; P=0.020) were independent predictors for poor
OS.
| Table IV.Cox multivariate prognostic analysis
for 41 patients with urothelial carcinoma of the bladder. |
Table IV.
Cox multivariate prognostic analysis
for 41 patients with urothelial carcinoma of the bladder.
|
|
|
|
| 95% CI for HR |
|---|
|
|
|
|
|
|
|---|
| Marker | Risk factor | P-value | HR | Lower | Upper |
|---|
| Sex | Male | 0.020 | 0.140 | 0.027 | 0.732 |
| CD147 | Positive | 0.022 | 6.889 | 1.315 | 36.084 |
| c-Fos | Positive | 0.033 | 4.636 | 1.128 | 19.057 |
| c-Jun | Positive | 0.029 | 4.589 | 1.172 | 17.968 |
Discussion
The present study demonstrated that increased
expression of c-Jun or CD147 proteins was predictive of poor OS for
UCB, although c-Fos protein was not significantly associated with
OS. Furthermore, both CD147 and c-Jun positive expression, or all
positive expression of c-Fos, c-Jun and CD147, served as an index
of poor OS for UCB. All above indicated that overexpression of
c-Jun and CD147, perhaps including c-Fos may contribute to tumor
progression of UCB.
Many genetic factors have been identified as being
associated with bladder cancer (19,20).
However, not much is understood regarding the molecular mechanisms
of its tumorigenesis and tumor progression. CD147 expressed by
tumor cells stimulates peritumoral fibroblasts to produce matrix
metalloproteinases, thus contributing to tumor invasion and
metastasis (21). Previous studies
have highlighted the pivotal role of CD147 protein in
carcinogenesis and tumor progression. CD147 expression in breast
carcinomas is associated with risk factors such as poor
histological grade, negative hormone status, the mitotic index and
tumor size (22). High CD147
immunostaining scores in hepatocellular carcinomas correlate
significantly with tumor grading and tumor-node-metastasis stages
(22). It is reported that CD147
is involved in a regulatory loop of miRNA and transcription factors
in breast cancer invasion and metastasis (23). In gastric carcinoma, CD147
expression is positively correlated with tumor size, depth of
invasion and lymphatic invasion, but not with lymph node
metastasis, staging, or differentiation (24). Previous studies of the authors have
demonstrated that CD147 may be involved in the progression of
prostate cancer and renal cell carcinoma, and can be used as an
independent prognostic factor of these cancers (21,25).
However, CD147 protein expression patterns within esophageal
squamous cell carcinoma and dysplastic lesions are not associated
with any of these clinicopathologic factors (26). In the present study, the results
indicated that CD147 was overexpressed in human bladder cancer.
However, intense expression of CD147 in bladder cancer was not
significantly correlated with the TNM stage, WHO grade and tumor
size (Table I), which disaccorded
with the results of Riethdorf et al (27). In addition, Riethdorf's study
identified that the expression levels of CD147 in invasive
transitional cell carcinomas of bladder were higher than those in
noninvasive tumors (27). These
discrepancies suggest that there are different clinical features of
CD147 expression in bladder cancer cells of different patients of
UCB, in consideration of all cases in the current study with no
recurrence and metastasis following transurethral resection and
partial or radical cystectomy therapy. It has already been
demonstrated that CD147 overexpression is associated with poorer
outcome and shorter survival time in patients with solid tumors
(28,29). The present findings supported this
hypothesis, as CD147 expression was associated with poor survival
in univariate analysis (Fig. 2).
Cumulatively, these results indicated that CD147 may be one of the
key molecular markers to identify high risk of progression in
bladder cancer, but other key molecules involved in tumor
progression such as transcription factors must be identified to
develop more effective therapeutic targets and to supply more
reliable judgments of the prognosis of UCB patients. The study of
Hagemann et al (30)
demonstrated that CD147 activates multiple transcription factors,
including AP-1 (c-Fos, c-Jun and FosB) and NF-κB in cardiomyocytes.
The AP-1 complex consists of two elements, c-Fos and c-Jun
(31). The AP-1 complex has been
implicated in the transformation and progression of cancer
(32). Although a few studies have
discovered some alternative activities of c-Jun like anti-cancer
property (33), most research
suggests that c-Jun contributes to tumor initiation and increased
invasiveness (18,34). In breast cancer, high expression
levels of c-Jun in MCF-7 cells can result in an overall increased
invasion by increased cellular motility and increased expression of
MMP-9 (35). The observed
phenotype for MCF-7 cells with c-Jun overexpression is similar to
that observed clinically in advanced breast cancer, which had
become hormone unresponsive (36).
The other most common element of the AP-1 complex, c-Fos, has also
been identified as independent predictor of poor survival in breast
cancer (14). CD147 has been
regarded as a prognostic marker for breast cancer with MMP-9
(37). It seems that there is a
reason to examine the hypothesis that c-Jun and CD147, and even
c-Fos, are involved in the progression of the human urothelial
carcinoma of the bladder, according to the present study results
and other previously-mentioned studies. However, future research
should aim to elucidate the mechanism of c-Jun, c-Fos and CD147 in
bladder cancer progression by enlarging sample size, and studying
in in vitro and in vivo models.
The present study indicated that increased
expression of the c-Jun and CD147 proteins, as well as
co-expression of CD147-c-Jun, c-Jun-c-Fos, or CD147-c-Jun-c-Fos has
prognostic significance for UCB patients. Therefore, high CD147 and
c-Jun expression may serve roles in UCB progression and may be
diagnostic and therapeutic targets in UCB whether alone or in
combination.
Acknowledgements
This work was supported by grants from National
Science and Technology Major Project (grant no. 2013ZX09301301) and
National High Technology Research and Development Program (grant
no. 2015CB553701).
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