Human Cancer Biology
Identification and Prognostic Significance of an
Epithelial-Mesenchymal Transition Expression
Profile in Human Bladder Tumors
Egbert Baumgart,2 Michael S. Cohen,2 Brasil Silva Neto,1,2 Micah A. Jacobs,2 ChadWotkowicz,2
Kimberly M. Rieger-Christ,1,2 Andreia Biolo,4 Ron Zeheb,3 Massimo Loda,5
John A. Libertino,2 and Ian C. Summerhayes1,2
Abstract
Purpose: Epithelial to mesenchymal transition (EMT) is reportedly an important transition in
cancer progression in which the underlying cellular changes have been identified mainly using
in vitro models. In this study, we examined the expression pattern of EMT markers in vivo and
determinedtheoccurrenceandclinicalsignificanceof these eventsinaseriesofbladdercarcinomas.
Experimental Design: Eight hundred and twenty-five tumor samples from 572 bladder cancer
patients were assembled in 10 tissue microarrays. Paraffin sections from each tissue microarray
were subjected to antigen retrieval and processed by immunohistochemistry for the expression
of E-cadherin, plakoglobin, h-catenin, N-cadherin, and vimentin.
Results: Pathologic expression of E-cadherin, h-catenin, plakoglobin, and vimentin were associated with the clinicopathologic variables of grade and stage with only the cytoplasmic localization of plakoglobin found associated with lymph node status. Associations between the
aforementioned markers were found significant as determined by the Spearman correlation coefficient with N-cadherin showing no associations in this analysis. In univariate survival analysis
involving patients who underwent cystectomy, the reduction or loss of plakoglobin significantly
influenced overall survival (P = 0.02) in which the median time to death was 2 years compared
with 4 years when a normal level of plakoglobin was recorded. When the analysis was done for
cancer-specific survival, low levels of both plakoglobin (P = 0.02) and h-catenin (P = 0.02)
significantly influenced survival.
Conclusion: The putative markers of EMTdefined within a panel of bladder carcinoma cell lines
were recorded in vivo, frequently associated with tumors of high grade and stage. Although multivariate analysis showed no significant influence of the EMT biomarkers on survival, alterations
associated with plakoglobin were identified as significant prognostic features in these tumors.
Epithelial to mesenchymal transition (EMT) is a process that
was first observed in embryonic development (1 – 3) and has
more recently been implicated as an underlying event in
neoplastic progression (4 – 7). To date, the definition and
occurrence of EMT in in vivo tumorigenesis remains
Authors’ Affiliations: 1Cell and Molecular Biology Laboratory, R.E. Wise M.D.
Research and Education Institute; Departments of 2Urology and 3Pathology, Lahey
Clinic, Burlington, Massachusetts ; 4 Department of Internal Medicine and
Cardiology, Hospital De Clinicas de Porto Alegre, Porto Alegre, Brazil; and
5
Department of Pathology, Dana-Farber Cancer Center, Boston, Massachusetts
Received 9/20/06; revised 12/18/06; accepted 1/10/07.
Grant support: R01-DK59400 (I.C. Summerhayes). B. Silva Neto and A. Biolo
were sponsored by Coordenacao deAperfeicoamento de Pessoal de Ensino Superior.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with18 U.S.C. Section1734 solely to indicate this fact.
Note: E. Baumgart, M.S. Cohen, and B. Silva Neto contributed equally to this work
and are listed in alphabetical order.
Requests for reprints: Ian C. Summerhayes, Cell and Molecular Biology
Laboratory, Robert E. Wise M.D. Research and Education Institute, Lahey Clinic,
31Mall Road, Burlington, MA 01805. Phone: 781-744-2990; Fax: 781-744-2984;
E-mail: Ian.C.Summerhayes@ lahey.org.
F 2007 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-06-2330
www.aacrjournals.org
controversial (6, 8); however, the conceptual framework
embracing loss of epithelial markers and gain of mesenchymal markers has been reportedly associated with numerous
cancers (9 – 11), often identified in cell lines established from
tumors representing different grades and stage (10, 12). In
this way, the discontinuous progression model observed
within a panel of cell lines of common tissue origin may be
closely linked to the differentiation status of cells. In most
cases, the projected transition between epithelial and mesenchymal phenotypes is accompanied by increased motility and
invasive potential.
With the advent of the establishment of in vitro models of
EMT, elicited by the action of different factors on alternative cell
types (13 – 18), the existence of EMT in progression becomes
more credible. In addition, such models enable us to confirm
events linked to EMT identified in discontinuous progression
models and to discover further molecular events underlying
this transition (7, 19). Alterations associated with the cadherin/
catenin complex often feature at the center of EMT related to
increased migration and invasion of cells (19). Such events will
follow different courses in different models dependent on the
expression profile of the cadherins in the targeted cell. In a
discontinuous model of EMT in bladder cancer, we have
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Human Cancer Biology
identified loss or reduced expression of E-cadherin accompanied by the silencing of plakoglobin expression as late-stage
events in bladder tumorigenesis (20). This change was linked to
increased motility and invasion potential. In bladder carcinoma
cell lines, loss of E-cadherin expression is accompanied by
novel expression of N-cadherin, representing cadherin switching from an epithelial-specific classic cadherin to a mesenchymal-specific cadherin (21). Such EMT-related events
accompany increased motility and invasion in bladder carcinoma cells. In a previous report, we have shown novel
expression of N-cadherin in human bladder tumor tissue
(22). In this study, using a tissue microarray format, we
evaluated five biomarkers linked to EMT to determine the
occurrence of expression of these molecules in vivo and the
potential prognostic value of such events within a well-defined
series of bladder carcinomas.
Materials and Methods
Cell culture. Human bladder cell lines RT4, RT112, HU456,
BC16.1, CUBIII, 5637, PSI, HT1197, HT1376, SCaBER, EJ, KK47, J82,
UM-UC-3, and TCCSUP were maintained in DMEM supplemented with
7.5% fetal bovine serum and penicillin/streptomycin.
Invasion assays. In vitro invasion assays were carried out using
modified Boyden chambers consisting of Transwell (8-Am pore size;
Corning Costar Corp., Cambridge, MA) membrane filter inserts in
24-well tissue culture plates. For invasion assays, the upper surfaces of
the membranes were coated with Matrigel (Collaborative Biomedical
Products, Bedford, MA) and placed into 24-well tissue culture plates
containing 600 AL of NIH/3T3 conditioned medium (experimental) or
plain DMEM (control). Cells (1 105) were added to each Transwell
chamber and allowed to invade toward the underside of the membrane
for 24 h at 37jC. Cells that pass through the membrane were fixed in
methanol, stained with crystal violet, and counted under a light
microscope.
Immunocytochemical staining of bladder carcinoma cell lines. Cells
were grown on glass slides, washed with PBS, and fixed in 3.7% formaldehyde for 15 min at room temperature. Cells were then rinsed in three
changes of PBS and permeabilized in 0.5% Triton X-100 in PBS. Following
three washes in PBS, cells were stained for EMT biomarkers using the
antibodies listed below. Immunostaining was done on an automated
stainer (Autostainer Plus, DAKO Corporation, Carpinteria, CA).
Western blot analysis. Subconfluent dishes of cells were washed in
PBS followed by lysis in hot sample buffer [2 ESB-0.08 mol/L Tris (pH
6.8); 0.07 mol/L SDS, 10% glycerol, 0.001% bromophenol blue, and 1
mmol/L CaCl2] and sheared through a 26-gauge needle. Lysates were
then assayed for protein concentration using the bovine serum albumin
method (Pierce, Rockford, IL). After determination of protein content,
h-mercaptoethanol (1%) was added to each sample. Samples were
boiled for 5 min, and protein was loaded in each lane of a 7.5% or
12.5% polyacrylamide gel. Proteins were transferred overnight onto
nitrocellulose. Membranes were blocked in 10% milk in TBS with
0.05% Tween and incubated with primary antibody overnight at 4jC.
Blots were washed in TBS with 0.05% Tween, thrice for 15 min each,
and incubated with secondary antibody linked to horseradish
peroxidase for 60 min at room temperature. Blots were then washed
as described above and developed with an enhanced chemiluminescence kit (Amersham, Arlington Heights, IL).
Clinical samples. We have used tumor tissue samples from patients
diagnosed with bladder cancer at the Lahey Clinic Medical Center
between 1990 and 2005 under an institutional review board – approved
protocol. Formalin-fixed, paraffin-embedded tumor tissues from
patients were retrieved from the archives of the Pathology Department.
The tumor stage was determined using tumor-node-metastasis classification and graded according to WHO guidelines.
Clin Cancer Res 2007;13(6) March 15, 2007
Construction and immunohistochemical staining of tissue microarrays. Eight hundred and twenty-five tumor samples from 572
patients were assembled in 10 tissue microarrays. All tumor samples
were transitional cell carcinomas. Tissue microarrays were designed
with replicas for each tumor sample and each control. Controls
included normal tissue from prostate, testis, tonsil, liver, cerebellum,
kidney, lung, and bladder. Strategic placement of control cores in each
tissue microarray enabled definitive orientation during the scoring of
the tissue microarrays. Each tissue microarray consisted of 400 tissue
cores (four cores per specimen) and control tissue cores for
immunohistochemical validation and orientation. Multiple 4-Am
sections were cut and stored at 4jC in the presence of a desiccant
before immunohistochemical staining. Individual tissue microarray
sections were deparaffinized and antigen retrieval in citrate buffer (pH
6.0; DAKO) was done. The antibodies were diluted with DAKO
antibody diluent solution. Immunohistochemical staining was done on
an automated stainer (Autostainer Plus, DAKO) using a high-sensitivity,
polymer-based detection system (EnVision, DAKO). A negative control
was included using a nonspecific mouse antibody solution (DAKO)
substituting for the primary antibody.
Antibodies. Mouse monoclonal antibodies to cytokeratin, E-cadherin (DAKO), h-catenin, and plakoglobin (BD Transduction Laboratories, San Diego, CA) were diluted 1:25; 1:100, and 1:100 (working
concentrations, 0.75, 7.56, and 2.5 Ag/mL), respectively, for use in
immunohistochemistry. Mouse monoclonal antibodies to mesenchymal cell markers N-cadherin (Zymed, San Francisco, CA) and vimentin
(DAKO) were diluted 1:100 (working concentration 5 Ag/mL) and 1:50
(working concentration 4.6 Ag/mL), respectively. The negative control
reagent (DAKO) is a cocktail of nonimmune mouse immunoglobulins
(IgG and IgM) and was purchased prediluted. The specificity of each
antibody in immunohistochemistry was determined using xenograft
sections derived from bladder cell lines in which the expression profile
of the antigen of interest had been previously identified.
Scoring of immunohistochemistry. The tissue sections were scored
semiquantitatively, assessing staining intensity and protein localization,
including membrane, cytoplasmic, or nuclear localization. In the case of
E-cadherin, h-catenin, and plakoglobin, a staining intensity scale of 0 to
3 was applied. In normal bladder tissue cores, the staining intensity with
each antibody was recorded as 3 with membrane localization throughout all epithelial layers of the mucosa. In tissue sections that displayed
heterogeneous staining throughout the section or between cores from
the same tumor, the worst-case-scenario score was assigned to that
sample when >5% of the tumor cells displayed this phenotype. Greater
than 90% concordance between scores from different cores of the same
tumor was recorded. In samples where scoring differed between cores,
the worst-case-scenario score was recorded for analysis. Specimens that
exhibited a complete absence of staining or faint staining in <5% cells
were scored negative. In the case of N-cadherin and vimentin, a positive
or negative score was given for each tissue sample because the presence
of either mesenchymal marker in bladder carcinoma cells represented
novel expression. Each tissue microarray was scored independently
(I.C.S. and M.L.). Where discordant results were obtained, both
individuals rereviewed the stained cores to obtain a consensus.
Statistical analysis. Comparisons of groups were done by Pearson’s
m2 test. Correlation between markers was determined by Spearman’s
correlation coefficient. Univariate overall and cancer-specific survival
analysis was done using the product-limit procedure (Kaplan-Meier
method), with the surgery date as the entry date. The log-rank (CoxMantel) test was used to compare survival curves for different categories
of each variable. Variables with effect on survival in univariate analyses
(P V 0.15) were examined by log-log plot to determine how these
variables could be incorporated into a Cox proportional hazards
regression models, and variables with P V 0.1 were maintained in the
model. Test for interactions were carried out for the variables that were
significantly related to survival in Cox regression analysis. Data were
analyzed using the SPSS software package, version 11.5. B. Silva Neto
and A. Biolo did the statistical analysis.
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The EMT Phenotype in BladderTumors
Table 1. Expression of EMT phenotype in bladder carcinoma cell lines
RT4
RT112
HU456
BC16.1
CUBIII
5637
PSI
HT1197
HT1376
SCaBER
EJ
KK47
J82
UM-UC-3
TCCSUP
E-cadherin
Plakoglobin*
B-catenin*
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+
+++
+
+
+
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
++
+++
++
++
++
N-cadherin
Vimentin
+
+
+
+
+
+
+
+
+
+
+
+
+
Morphology
Invasionc
E
E
E/M
E
E
E
E
E
E
E
M
E/M
M
M
M
+
+
+
+
+
+
+
+++
++
+
NOTE: +++, expressed at high levels; ++, expressed at moderate levels; +, expressed at low levels; , no detectable expression; E, epithelial
morphology; E/M, intermediate morphology between epithelial and mesenchymal cells; M, mesenchymal morphology.
*No evidence of nuclear localization of catenins was recorded by immunocytochemistry.
cInvasion was assessed over a 16-h period in an in vitro assay. Invasion assay results were scored as number of cells that had traversed the
membrane per 1 mm grid; , 0 to 4 cells; +, 5 to 20 cells; ++, 21 to 50 cells; +++, >51 cells.
Results
Characterization of EMT markers in bladder carcinoma
cells. To establish the occurrence of the putative EMT
phenotype in bladder carcinoma cells, we initially screened a
panel of 15 cell lines for the expression of the five biomarkers
we propose to evaluate in tissue sections. Table 1 shows the
expression profile of each component as determined in
Western blot analysis and includes morphologic and in vitro
invasion data for each cell line. No nuclear localization of
proteins was detected in cell lines using immunocytochemistry
(data not shown). Morphologic classification of epithelial
morphology was defined by tightly adherent cuboidal cells
growing as discrete colonies with mesenchymal morphology
Table 2. Histologic and clinical variables of arrayed transitional cell carcinomas of the bladder
n
Pathologic stage
pTa
pTis
pT1
pT2-T4
Tx
Grade
1/2
3
Stage/grade
Ta/G1-G2
TaG3
T 1 G2
T 1 G3
T2-T4/G2
T2-T4/G3
Tx/G1-G2
Tx/G3
N/M stage
N+
M+
Vascular invasion, yesF
Sex, male
Smoking history, yes
Mean age at cystectomy
Mean follow-up for survivors (y)
n
Samples (%)
825
317 (38.4)
40 (4.8)
92 (11.1)
321 (39)
55 (6.7)
(36.9)
(4.2)
(9.4)
(46.9)
(2.6)
572
298 (36.2)
526 (63.8)
n
10
22
32
233
2
572
248 (30.1)
69 (8.4)
17 (2.1)
74 (9)
25 (3)
296 (35.9)
7 (0.9)
48 (5.8)
27 (9)
272 (91)
299
167 (29.2)
44 (7.7)
11 (1.9)
43 (7.5)
18 (3.1)
250 (43.8)
1 (0.2)
14 (2.4)
83 (29.3)
33 (8.4)
203 (45.4)
262
317
325
572
543
(3.3)
(7.4)
(10.7)
(77.9)
(0.7)
299
198 (34.6)
374 (65.4)
824
Cystectomy (%)
299
211
24
54
268
15
824
283
393
447
Patients (%)
572
78 (29.8)
30 (9.5)
166 (51.1)
438 (76.6)
393 (72.4)
6 (2)
4 (1.3)
6 (2)
26 (8.7)
15 (5)
218 (72.9)
0
2 (0.7)
240
270
265
299
282
62 (25.8)
8 (3.0)
136 (51.3)
231 (77.3)
209 (74.1)
67.1 F 9.7
5.1 F 3.3
NOTE: Values in table are expressed as number of samples or patients (%) or mean F SD. FTa and Tis were not considered in the analysis. All Tis
are grade 3.
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Human Cancer Biology
Table 3. Associations between EMT markers and clinicopathologic factors in patients with bladder cancer
P
Low
B-catenin
P
37.7 (427)
60.2 (304)
<0.001
16.7 (426)
47.6 (313)
<0.001
36 (292)
54.3 (490)
<0.001
12.5 (289)
40.4 (502)
51.9 (231)
60.8 (194)
0.07
52.7 (182)
61.5 (78)
0.19
Low
E-cadherin
Tumor stage
Superficial
Invasive
Grade
1/2
3
Vascular invasion
No
Yes
Node stage
Negative
Positive
P
Vimentin
P
15.2 (429)
57.9 (309)
<0.001
6.8 (427)
30.9 (314)
<0.001
<0.001
15 (287)
44.3 (503)
<0.001
6.2 (292)
24.7 (502)
<0.001
41.1 (236)
47.2 (199)
0.2
50.2 (233)
52.3 (199)
0.671
27.5 (236)
30.5 (200)
0.5
42.6 (190)
52.5 (80)
0.14
52.4 (191)
60.5 (81)
0.218
26.6 (192)
31.7 (82)
0.4
Low
plakoglobin
NOTE: Values in table are expressed as percentages (number of samples).
defined by poorly adherent carcinoma cells displaying a stellate
morphology. From the aforementioned data, it is clear that the
projected EMT phenotype is recorded within this panel of
bladder carcinoma cell lines and is correlated with a mesenchymal morphology and invasive phenotype as assessed in an
in vitro assay.
EMT phenotype in bladder tumor tissue. If the EMT phenotype identified in the bladder carcinoma cell panel is a valid
paradigm in bladder cancer, we would expect to see the
proposed modulations in epithelial and mesenchymal cell
markers associated with more aggressive bladder disease. To test
this postulate, E-cadherin, plakoglobin, h-catenin, N-cadherin,
and vimentin expression levels and localization were assessed
in tissue microarrays of primary bladder tumors. The construction of 10 microarrays included 825 transitional cell carcinoma
samples from 572 patients. The tumor-node-metastasis and
histologic grade of the samples is shown in Table 2. Analysis by
number of patients as opposed to number of samples revealed
no difference in the results. In this study, the analysis of the
superficial tumor group included pTa, pTis, and pT1 with the
invasive group represented by pT2-T4 tumors. When T1G3 was
included in the invasive group, the results did not change (data
not shown).
E-cadherin expression. A reduction or loss of E-cadherin
expression was significantly correlated with pathologic tumor
stage (P < 0.001), histologic grade (P < 0.001), but not lymph
node involvement or lymphovascular invasion in bladder
carcinomas. Loss or reduction in E-cadherin expression was
recorded in 37.7% of superficial tumors and 60.2% of
invasive tumors with similar values recorded for cytoplasmic
localization of E-cadherin, 26% and 58.1%, respectively
(Table 3).
b-Catenin expression. A reduction or loss of expression, in
addition to a cytoplasmic localization of h-catenin, showed
significant correlation with tumor grade and stage (P < 0.001)
in bladder carcinomas. Neither the intensity nor the localization was found associated with a positive lymph node status or
vascular invasion (Table 3). A reduction or loss of h-catenin
expression was recorded in 16.7% of superficial tumors and
47.6% of invasive tumors (Table 3). The cytoplasmic localization of h-catenin revealed a greater differential between
Clin Cancer Res 2007;13(6) March 15, 2007
superficial and invasive tumor groups, 17.9% and 58%,
respectively. Nuclear localization of h-catenin was recorded in
27 of 709 (3.8%) patient tumors, including 1 Ta, 9 T1, and 17
T2-T4 bladder tumors.
Plakoglobin expression. A reduction or loss of expression
of plakoglobin and the cytoplasmic localization of plakoglobin was significantly associated with tumor grade and stage
(P < 0.001). In addition, plakoglobin localization was also
correlated with nodal status in bladder cancer patients (P =
0.04). Neither the level of plakoglobin expression nor the
location was associated with vascular invasion. Loss or
reduced plakoglobin expression was recorded in 15.2% of
superficial tumors and 57.9% of invasive tumors. As with
h-catenin localization, there existed a marked differential
between the frequency of cytoplasmic localization of
plakoglobin in the superficial and invasive tumor groups,
23.1% and 72.1%, respectively (Table 3). Nuclear localization of plakoglobin was recorded in 13 of 726 (1.8%)
bladder carcinomas, including four Ta, four T1, and five T2-T4
lesions.
N-cadherin expression. Novel expression of N-cadherin was
recorded in 61 of 746 (8.2%) bladder carcinomas (Fig. 1A and
B), including 32 superficial and 24 invasive tumors with 5
tumors that remained unstaged. Membranous N-cadherin
staining was rarely recorded expressed throughout a tumor
(Fig. 1B) but displayed a limited focal localization (Fig. 1A). Of
the 61 tumor samples positive for N-cadherin expression, only
21 were scored as positive in all scorable tumor cores from a
single patient. Novel expression of N-cadherin did not show a
significant association with grade, stage, lymph node involvement, or vascular invasion.
Vimentin expression. Novel expression of vimentin in
bladder carcinoma cells in vivo was recorded in 143 of 795
(18%) tumors, including 29 superficial and 97 invasive lesions
with 17 tumors that remained unstaged. Expression of vimentin
(Fig. 1C and D) was significantly associated with tumor grade
and stage (<0.001) but not with nodal involvement or vascular
invasion. Figure 2 shows immunostaining for all markers in the
same tissue core, demonstrating coexpression of the epithelial
and mesenchymal intermediate filament proteins keratin
(Fig. 2A) and vimentin (Fig. 2E), respectively.
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The EMT Phenotype in BladderTumors
Table 3. Associations between EMT markers and clinicopathologic factors in patients with bladder cancer
(Cont’d)
N-cadherin
P
E-cadherin cytoplasmic
location
P
B-Catenin cytoplasmic
location
P
Plakoglobin cytoplasmic
location
P
8.1 (393)
8.0 (301)
0.94
26 (358)
58.1 (172)
<0.001
17.9 (407)
58.0 (257)
<0.001
23.1 (416)
72.1 (265)
<0.001
10.6 (263)
6.8 (482)
0.07
19.3 (254)
51.3 (308)
<0.001
13.2 (280)
47.7 (428)
<0.001
18.5 (281)
57.7 (444)
<0.001
9.2 (228)
5.7 (193)
0.18
51.8 (137)
57.1 (112)
0.40
53.9 (206)
57.0 (158)
0.56
62.9 (202)
68.2 (170)
0.28
6.0 (184)
11.3 (80)
0.14
49 (98)
59 (39)
0.29
56.9 (160)
67.8 (59)
0.14
65.0 (160)
78.9 (71)
0.04
Associations among EMT markers. Loss or reduced expression of E-cadherin was significantly associated with low
h-catenin (P < 0.01; Spearman correlation coefficient, 0.446),
low plakoglobin (P < 0.01; Spearman correlation coefficient,
0.370), and novel expression of vimentin (P < 0.01; Spearman
correlation coefficient, 0.109). Low expression of h-catenin
was independently associated with low plakoglobin expression
(P < 0.01; Spearman correlation coefficient, 0.441) and
vimentin expression (P < 0.01; Spearman correlation coefficient, 0.181). In turn, low plakoglobin was found significantly associated with vimentin expression (P < 0.01; Spearman
correlation coefficient, 0.231). No significant associations
were found with any of the aforementioned EMT markers and
N-cadherin.
Survival analysis. Because there was no complete follow-up
data for the superficial group, survival analysis was confined to
the cystectomy patient group. Univariate analysis involving each
of the proposed EMT markers in overall survival showed that the
reduction or loss of plakoglobin expression (P = 0.02) was
significantly associated with patient survival with a trend for
association with h-catenin (P = 0.11; Fig. 3A and C). In contrast,
no significant difference was recorded for E-cadherin or Ncadherin levels of expression (Fig. 3E and G). Within this patient
group, the clinical variables of T-stage, lymph node status, and
vascular invasion showed a significant correlation with survival,
as previously established. The 5-year survival rate for patients
whose tumors displayed a loss or reduction in expression of
plakoglobin was 29.4% compared with 41.5% when plakoglobin displayed normal levels of expression. Ten-year survival
rates were 22.9% and 29.2%, respectively, when the same
plakoglobin variables were analyzed. The median time to death
for patients with loss or reduced plakoglobin expression was 2
years compared with 4 years when a normal plakoglobin level
was recorded. The 5-year survival rate for patients whose tumors
displayed a loss or reduction in expression of h-catenin was
29.1% compared with 40.8% when h-catenin displayed normal
levels of membrane staining. Ten-year survival rates were 24.2%
and 23.5%, respectively, when the same h-catenin variables
were analyzed. The median time to death for loss or reduced hcatenin was 2.4 years compared with 3.8 years when h-catenin
displayed high expression.
When the outcome was limited to cancer-specific survival,
the loss or reduction in plakoglobin expression (P = 0.02)
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and the reduction or loss of expression of h-catenin (P =
0.02) were significant for survival (Fig. 3B and D). As
expected, T-stage, lymph node status, and vascular invasion
correlated with cancer-specific survival. The 5- and 10-year
survival rates were 43.7% and 41%, respectively, for loss or
reduction of plakoglobin compared with 55% and 52.7%,
respectively, in the group with normal levels of expression. With
h-catenin, 5- and 10-year survival rates were 41.9% and 39.4%,
respectively, when a loss or reduction of expression was
recorded compared with 56.3% and 53.7%, respectively, for
normal levels of expression. E-cadherin and N-cadherin did not
show a significant difference for cancer-specific survival (Fig. 3F
and H).
Multivariate analysis was done using plakoglobin, h-catenin,
tumor stage, age, and vascular invasion because all showed
significance in univariate analysis as recorded in Tables 4 and 5.
For overall survival, vascular invasion and age of diagnosis were
strong independent prognostic predictors (hazard ratio; 1.83;
P < 0.001 and hazard ratio; 1.03 per year increment, P = 0.006,
respectively). Loss of plakoglobin had a hazard ratio of 1.31
(P = 0.11), and h-catenin was no longer associated with
survival (P = 0.82).
For cancer-specific survival, plakoglobin and h-catenin sites
were included in the model with the aforementioned variables.
Vascular invasion was the only significant independent
predictor in the model (hazard ratio, 2.57; P < 0.001).
Discussion
In this study, we have assessed the expression and prognostic
value of a panel of five markers associated with the EMT
phenotype. Loss of E-cadherin and novel expression of Ncadherin represent defining features of EMT, both of which
have been reported in bladder tumorigenesis (22, 23). Linked
to this transition has been the expression of the mesenchymal
intermediate filament protein vimentin, often accompanied by
altered cytokeratin expression (19). In addition, delocalization
of h-catenin has been implicated in the EMT process (9).
Numerous studies have shown loss or reduced expression of
plakoglobin in epithelial cells in the absence of E-cadherin
expression and more recently this phenotypic change has been
implicated as part of the EMT phenotype (24). Changes in the
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Fig. 1. Immunohistochemical staining
showing N-cadherin (A and B) and
vimentin (C and D) in low (A and C) and
high-grade (B and D) bladder tumors.
A, novel expression of N-cadherin in a
superficial tumor showing focal localization
within the tumor. B, N-cadherin expression
throughout an invasive bladder tumor.
C, absence of vimentin expression in
epithelial cells of a superficial bladder tumor.
D, novel vimentin expression in carcinoma
cells of an invasive bladder tumor.
aforementioned proteins represent EMT end points in which
the expression of each may be modulated by a growing number
of molecules involved in transcriptional regulation, cell
signaling, and modulation of the tumor microenvironment
(for review, see refs. 7, 14, 19, 24). However, there remains
some controversy over the occurrence of such events in vivo
originating from the apparent rarity of the EMT-like morphologic changes observed by pathologists in primary tumor
sections and secondary lesions (8).
The counter argument to this necessarily requires recording
EMT-related events in vivo during tumor progression (25, 26).
Evidence for such events is beginning to emerge and presently
include reports of Snail 1 protein, avh6 integrin, and nuclear
h-catenin visualized at the invading edge of colon cancer
tumors (14, 19). A search for the expression of such EMTrelated proteins may be best served from the analysis of total
tumor sections because the reported limited localization of
proteins to the invasive front of the tumor may be missed in
alternative tissue sampling formats.
A reduction or loss in expression of E-cadherin has long been
recognized as an important primary event in bladder tumorigenesis often linked to poor prognosis (23, 27 – 29). In this
study, altered E-cadherin expression was significantly associated
with low h-catenin and plakoglobin expression along with
novel expression of vimentin. Consideration of changes
Clin Cancer Res 2007;13(6) March 15, 2007
associated with the cadherin complex proteins revealed a
significant difference between the frequency of such events in
the superficial and invasive tumor groups, linking such changes
to tumor progression. Although altered expression or localization of E-cadherin were not found associated with lymph node
involvement or vascular invasion, the cytoplasmic localization
of plakoglobin was a significant indicator of lymph node
involvement.
Nuclear localization of h-catenin and plakoglobin was
recorded in 3.8% and 1.8% of tumor samples, respectively,
and was always observed in the presence of cytoplasmic
localization of the catenin member. The reported frequency
of nuclear localization of h-catenin in bladder tumors ranges
between 0% and 22% identified in study groups involving
small patient numbers (30 – 33). Mutations associated with
h-catenin have been shown to result in the nuclear localization
of this catenin family member in bladder tumors (30). Nuclear
localization of plakoglobin seems to be a less frequent finding
and although nuclear plakoglobin has been reported in other
cancers (34, 35), we are not aware of any reports of this finding
in bladder tumors. The nuclear localization of either h-catenin
or plakoglobin was observed throughout the tumor section in a
majority of samples whereby isolated islands of cells representing <5% of tumor cells with apparent nuclear staining were not
scored as positive in this study. Given the potential putative
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The EMT Phenotype in BladderTumors
transient nature of EMT and the limited, possibly temporary,
nuclear relocalization of catenins associated with the invasive
edge of the tumor, we may have overlooked more minor
representation of EMT events.
Novel expression of N-cadherin has also been reported in
human bladder tumors (22) and was previously recorded in
39% (20 of 51) of tumors. In this more extensive study, we
have recorded N-cadherin expression in only 8.2% (61 of 746)
of bladder tumors using the same antibody and antigen
retrieval technique. Consistent with our previous report, novel
expression of N-cadherin was observed to be focal in nature
throughout the tissue microarray, and although the presence of
four cores of each tissue is calculated to be representative of the
tumor phenotype we believe that the N-cadherin expression
recorded in this study is an underrepresentation of the
frequency of expression in bladder tumors. Support for this
interpretation is found in the scores for each of the four cores
from a single tumor. A review of the results revealed detection
of N-cadherin expression in all of the cores from a single tumor
in approximately one third of the cases (21 of 61). It is also of
interest that N-cadherin expression was recorded approximately
equally in the superficial and invasive tumor groups. Moreover,
in one recent publication, N-cadherin was identified as a
prognostic marker of progression in superficial urothelial
tumors (36). Although we have shown that novel expression
of N-cadherin in bladder carcinoma cells promotes invasion
(37), the expression of N-cadherin throughout different tumor
grades and stage makes identification of additional functions a
necessary consideration.
Novel vimentin expression, the second mesenchymal
marker linked to EMT, was frequently recorded in the bladder
carcinoma cell lines that displayed the more stellate, fibroblast
morphology. However, past experience has revealed that
normal urothelium can express vimentin after an extended
period of in vitro culture (38). In this study, we have shown
novel expression of vimentin in bladder carcinoma cells
in vivo mainly associated with invasive bladder lesions.
Confirmation of the epithelial origin of vimentin-positive
cells was established in neighboring sections stained for total
keratin demonstrating coexpression of these two intermediate
filament types. No association was found between expression
of the two mesenchymal markers, N-cadherin and vimentin.
Whereas N-cadherin was found not to be associated with any
of the clinical variables studied, vimentin expression was
significantly associated with tumor grade and stage. In
addition, novel vimentin expression showed significant
association with reduced expression of both h-catenin and
plakoglobin. Expression of vimentin was recorded in 18%
Fig. 2. Immunohistochemical staining of
different sections from a single tissue
microarray showing the same tumor core
incubated with antibodies to keratin (A),
E-cadherin (B), h-catenin (C), plakoglobin
(D), vimentin (E), and N-cadherin (F).
Note coexpression of intermediate filaments
keratin and vimentin (compare A and E) in
the absence of N-cadherin expression (F).
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Fig. 3. Kaplan-Meier curves among patients with bladder cancer after radical cystectomy. Plakoglobin expressionBoverall (A) and cancer-specific (B) survival. h-Catenin
expressionBoverall (C) and cancer-specific (D) survival. E-cadherin expressionBoverall (E) and cancer-specific (F) survival. N-cadherin expressionBoverall (G) and
cancer-specific (H) survival, respectively.
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The EMT Phenotype in BladderTumors
(143 of 795) of bladder tumors, establishing novel expression
of this intermediate filament in vivo. It is interesting that a
subgroup of superficial tumors (6.8%) was identified that
displayed novel expression of vimentin, where, given the
association found with tumor stage and vimentin in invasive
tumors, it is desirable to establish whether this marker
identifies superficial tumors at heightened risk to progress as
has been reported for N-cadherin (36).
In this study, survival data involved only patients that
underwent cystectomy. For overall survival, the reduction or
loss of plakoglobin was the one putative EMT variable that
showed significant association with patient survival. As
expected within this group of patients, the clinical variables
of tumor stage, lymph node status, and vascular invasion
also showed a correlation with survival. The median time to
death for patients with loss or reduced plakoglobin expression was half that (2 years) recorded for patients with high
expression of plakoglobin (4 years). Indeed, a previous study
within a small group of bladder cancer patients (39) also
reported that altered plakoglobin expression correlated with
poor survival. In addition, alterations in plakoglobin expression have recently been identified as a marker of progression in T1 bladder tumors (40). A similar pattern of loss
or reduced expression was recorded for h-catenin, although
Table 4. Univariate Cox regression analysis
Variable
Overall survival
Vascular invasion
Age (1-y increment)
Sex (male)
Smoke
Tumor stage (T2-T4)
Grade 3
Lymph node (+)
Metastasis (+)
Plakoglobin (low expression)
Plakoglobin (cytoplasm)
h-catenin (low expression)
h-catenin (cytoplasm)
E-cadherin (low expression)
E-cadherin (cytoplasm)
N-cadherin (novel expression)
Vimentin (novel expression)
Cancer-specific survival
Vascular invasion
Age (1-y increment)
Sex (male)
Smoke
Tumor stage (T2-T4)
Grade 3
Lymph node (+)
Metastasis (+)
Plakoglobin (low expression)
Plakoglobin (cytoplasm)
h-Catenin (low expression)
h-Catenin (cytoplasm)
E-cadherin (low expression)
E-cadherin (cytoplasm)
N-cadherin (novel expression)
Vimentin (novel expression)
HR (95% CI)
P
1.87 (1.37-2.56)
1.03 (1.01-1.05)
0.83 (0.60-1.14)
1.04 (0.74-1.45)
2.23 (1.50-3.32)
1.21 (0.77-1.91)
1.71 (1.19-2.46)
2.31(1.08-4.97)
1.41 (1.05-1.89)
1.25 (0.88-1.76)
1.27 (0.95-1.70)
1.06 (0.76-1.48)
1.04 (0.77-1.41)
1.08 (0.72-1.59)
0.98 (0.43-2.22)
1.03 (0.73-1.46)
<0.001
<0.001
0.25
0.82
<0.001
0.41
0.003
0.03
0.02
0.21
0.11
0.75
0.78
0.71
0.96
0.85
2.48
1.02
0.75
0.96
3.35
1.15
2.15
3.21
1.54
1.39
1.55
1.47
1.32
0.93
1.20
1.24
<0.001
0.08
0.16
0.84
<0.001
0.63
<0.001
0.06
0.02
0.14
0.02
0.08
0.16
0.76
0.70
0.37
(1.68-3.66)
(0.99-1.04)
(0.51-1.11)
(0.64-1.44)
(1.88-5.96)
(0.66-2.00)
(1.42-3.24)
(1.40-7.36)
(1.07-2.22)
(0.90-2.16)
(1.08-2.24)
(0.95-2.27)
(0.90-1.92)
(0.56-1.52)
(0.49-2.93)
(0.80-1.92)
Abbreviations: HR, hazard ratio; 95% CI, 95% confidence interval.
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Table 5. Cox proportional-hazards regression
model—multivariate analysis
Variable
Overall survival
Vascular invasion
Age (1-y increment)
Tumor stage (T2-T4)
Plakoglobin (low expression)
h-catenin (low expression)
Cancer-specific survival
Vascular invasion
Age (1-y increment)
Tumor stage (T2-T4)
Plakoglobin (low expression)
Plakoglobin (cytoplasm)
h-Catenin (low expression)
h-Catenin (cytoplasm)
HR (95% CI)
P
1.83
1.03
1.45
1.31
1.03
(1.31-2.57)
(1.01-1.04)
(0.78-2.70)
(0.94-1.85)
(0.75-1.44)
<0.001
0.006
0.24
0.11
0.82
2.57
1.02
0.97
1.30
1.41
1.19
0.85
(1.51-4.35)
(0.99-1.05)
(0.40-2.38)
(0.77-2.21)
(0.75-2.63)
(0.60-2.23)
(0.42-1.70)
<0.001
0.14
0.95
0.33
0.28
0.60
0.65
NOTE: Variables initially included in the model were vascular
invasion, age, tumor stage, lymph node and metastasis status,
plakoglobin expression, and h-catenin expression. Plakoglobin and
h-catenin location were included only for cancer-specific survival
analysis.
such differences did not reach statistical significance. When
the outcome was limited to cancer-specific survival, both
the expression level and delocalization of plakoglobin and
h-catenin were found to have prognostic significance for
survival.
In this study, we have identified changes linked to EMT in
different models as events that occur in bladder tumors
in vivo. Loss of E-cadherin in bladder tumorigenesis is well
established and, more recently, we have shown novel
expression of N-cadherin in human bladder tumors (22).
However, reduced expression of plakoglobin associated with
these cadherin events has only recently been linked to EMT
(24). This combination of phenotypic changes has been
observed in all bladder carcinoma cell lines that display the
morphologic EMT phenotype accompanied by the enhancement of migration and invasion. In this study, we have shown
that loss of E-cadherin expression is significantly associated
with low plakoglobin expression even in the presence of Ncadherin expression. In addition, novel expression of vimentin
was also found significantly associated with alterations of Ecadherin, h-catenin, and plakoglobin expression. The lack of
association of N-cadherin with variables assessed as part of the
EMT phenotype is surprising, although, as previously stated,
we believe there is an underestimation of the frequency of
novel expression of N-cadherin. However, despite this caveat,
we have established the in vivo expression of putative EMT
alterations in bladder cancer in which loss or reduced
plakoglobin expression presented as a significant prognostic
feature in these tumors.
Acknowledgments
We thank Barbara Rothman for the technical help in immunohistochemical staining of tissue microarrays, and Robert Kelley and MatthewWszolek for help in collecting materials and data.
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Identification and Prognostic Significance of an
Epithelial-Mesenchymal Transition Expression Profile in
Human Bladder Tumors
Egbert Baumgart, Michael S. Cohen, Brasil Silva Neto, et al.
Clin Cancer Res 2007;13:1685-1694.
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