3064 Vol. 10, 3064 –3068, May 1, 2004
Clinical Cancer Research
Loss of 14-3-3␴ in Prostate Cancer and Its Precursors
Liang Cheng,1,2 Chong-Xian Pan,5
Jian-Ting Zhang,3 Shaobo Zhang,1
Michael S. Kinch,6 Lang Li,4 Lee Ann Baldridge,1
Christopher Wade,1 Zhiqiang Hu,3
Michael O. Koch,2 Thomas M. Ulbright,1 and
John N. Eble1
Departments of 1Pathology and Laboratory Medicine, 2Urology,
3
Pharmacology and Toxicology; the 4Division of Biostatistics; and the
5
University of Indiana Cancer Center, Indiana University School of
Medicine, Indianapolis, Indiana, and 6MedImmune Inc., Gaithersburg,
Maryland
ABSTRACT
Purpose: The 14-3-3 family proteins are highly conserved over many mammalian species. The ␴ isoform (also
called HME-1 or stratifin) is expressed in epithelial cells.
Loss of 14-3-3␴ is associated with failure to arrest the cell
cycle at the G2-M phase checkpoint after DNA damage that
leads to increased G2-type chromosomal aberrations. The
role of 14-3-3␴ in prostatic carcinogenesis is uncertain.
Experimental Design: We studied one hundred and
eleven specimens of invasive prostate adenocarcinoma with
paired, adjacent high-grade prostatic intraepithelial neoplasia and normal prostate epithelium. Immunohistochemistry was used to detect the expression of 14-3-3␴. The
findings were correlated with various clinical pathological
parameters.
Results: 14-3-3␴ is ubiquitously expressed at high levels
in normal prostate epithelium. Its expression is significantly
decreased in prostatic intraepithelial neoplasia and prostatic
adenocarcinoma. Ninety percent of samples of prostatic intraepithelial neoplasia had no or low 14-3-3␴ expression.
Ninety-seven percent of invasive adenocarcinomas had no
or low 14-3-3␴ expression. In most specimens (90%), suppression of 14-3-3␴ expression occurred during the development of prostatic intraepithelial neoplasia from normal
epithelium.
Conclusions: Our data suggest that loss of 14-3-3␴
contributes to the development of prostate adenocarcinoma.
14-3-3␴ expression is significantly decreased during the progression of normal prostatic epithelium to prostatic intraepithelial neoplasia and invasive cancer.
Received 11/28/03; revised 1/12/04; accepted 1/20/04.
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 with 18 U.S.C. Section 1734 solely to
indicate this fact.
Requests for reprints: Liang Cheng, Department of Pathology and
Laboratory Medicine, Indiana University Medical Center, University
Hospital 3465, 550 North University Blvd., Indianapolis, IN 46202.
Phone: (317) 274-1756; Fax: (317) 274-5346; E-mail: [email protected].
INTRODUCTION
The 14-3-3 family of proteins consists of seven isoforms,
which are highly conserved over many eukaryotic organisms (1,
2). Much recent interest has focused on the ␴ isoform (also
called HME-1 or stratifin), which is expressed in human epithelial cells (3–5). A crucial role of 14-3-3␴ is its control of the
G2 cell cycle checkpoint (6). At G2 phase, cdc2-cyclin B1
normally enters the nucleus to initiate mitosis. In response to
DNA damage, 14-3-3␴ is induced in a p53-dependent manner
and prevents the cdc2-cyclin B1 complex from entering the
nucleus. These changes provide an opportunity for DNA repair
of damage before further cell cycle progression (6, 7). Indeed,
cells lacking 14-3-3␴ function have impaired cell cycle control
after DNA damage and increased genomic instability. Suppression of 14-3-3␴ expression has been documented in transformed
cell lines, including v-Ha-ras (ras)-transformed mammary cells
(5), SV40-transformed human keratinocytes (8), and several
cancers (9 –12). Loss of 14-3-3␴ expression is caused by DNA
hypermethylation rather than gene deletion or mutation (9 –13)
and importantly, restoration of 14-3-3␴ expression by the DNA
demethylation agents (e.g., 5-aza-2⬘-deoxycytidine) normalize
control of the cell cycle (11).
To determine the role of 14-3-3␴ in prostate cancer, we
examined its expression in primary invasive prostate adenocarcinoma, high-grade prostatic intraepithelial neoplasia (PIN), and
in adjacent normal prostate epithelium.
MATERIALS AND METHODS
Patients. Radical prostatectomy specimens (n ⫽ 111)
containing invasive prostatic adenocarcinoma with adjacent
high-grade PIN and normal prostate epithelium were obtained
from the surgical pathology files of Indiana University Medical
Center from 1990 to 1996. These cases were selected to represent the full spectrum of Gleason grade and pathological stages.
The patients ranged in age from 44 to 77 years (mean ⫽ 63
years). Grading of the primary tumor from radical prostatectomy
specimens was performed according to the Gleason system (14).
The Gleason scores ranged from 4 to 10. Pathological staging
was performed according to the 1997 Tumor, Lymph Nodes,
and Metastasis System (15). The final pathological stages included T2a (11 patients), T2b (46 patients), T3a (33 patients),
and T3b (21 patients). Seventeen patients had lymph node
metastases at the time of surgery. This research was approved by
the Indiana University Institutional Review Board.
Generation of 14-3-3␴ Antibody. Goat polyclonal antibody specific to 14-3-3␴ was generated by immunizing goat
with a peptide mapping near the NH2 terminus of 14-3-3␴ of
human origin. The antisera was affinity purified. The specificity
of the purified IgG antibody was confirmed by Western blotting
and immunoprecipitation assay. No cross-reactivity with other
14-3-3 isoforms was observed.
Immunohistochemistry. Serial 5 ␮m-thick sections prepared from formalin-fixed, paraffin-embedded slices of prostate
adenocarcinoma specimens were used for the study. Tissue blocks
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Clinical Cancer Research 3065
that contained the maximum amount of tumor and highest Gleason
score were selected for each case. One representative slide from
each case was analyzed. We recognized the limitation of sample
variation. Slides were deparaffinized in xylene twice for 5 min and
rehydrated through graded ethanol solutions to distilled water.
Antigen retrieval was carried out by heating sections in 1 mm of
EDTA (pH 8.0) for 30 min. Endogenous peroxidase activity was
inactivated by incubation in 3% H2O2 for 15 min. Nonspecific
binding sites were blocked using Protein Block (DAKO Corp) for
20 min. Tissue sections were then incubated with the goat polyclonal antibody against human 14-3-3␴ (1:200 dilution, Santa Cruz
Biotechnology, Santa Cruz, CA) at 4°C overnight, followed by
incubation with biotinylated donkey antigoat antibody, and peroxidase-labeled streptavidin. Diaminobenzidine was used as the chromogen in the presence of hydrogen peroxide. Positive and negative
controls were run in parallel with each batch and demonstrated that
the procedure functioned properly.
Evaluation of 14-3-3␴ Expression. The extent and intensity of immunoreactivity for 14-3-3␴ were evaluated in benign epithelium, PIN, and adenocarcinoma from the same slide
for each case. Microscopic fields with the highest degree of
Table 1
RESULTS
Patient characteristics were illustrated in Table 1. Significant differences in 14-3-3␴ immunoreactivity distinguished normal prostate epithelium, PIN, and prostate adenocarcinoma. In
Patient characteristics and 14-3-3␴ staining percentage and intensity in prostate cancer
Patient characteristic
Primary Gleason grade
2
3
4
5
Secondary Gleason grade
2
3
4
5
Gleason sum
⬍7
7
⬎7
T classification
T2a
T2b
T3a
T3b
Lymph-node metastasis
Positive
Negative
Extraprostatic extension
Positive
Negative
Surgical margin
Positive
Negative
Vascular invasion
Positive
Negative
Perineural invasion
Positive
Negative
High-grade PINa
Positive
Negative
a
immunoreactivity were chosen for analysis. At least 1000 cells
were analyzed in each case. The percentage of cells exhibiting
staining in each case was evaluated semiquantitatively on a 5%
incremental scale ranging from 0 to 95%. A numeric intensity
score was set from 0 to 3 (0, no staining; 1, weak staining; 2,
moderate staining; and 3, strong staining). These methods were
described previously (16 –18).
Statistical Analysis. The mean percentage of immunoreactive cells in benign epithelium, high-grade PIN, and adenocarcinoma were compared using the Wilcoxon-paired signed
rank test. The intensities of staining for 14-3-3␴ in benign
epithelium, high-grade PIN, and adenocarcinoma were compared using Cochran-Mantel-Haenszel tests for correlated ordered categorical data. A P ⬍ 0.05 was considered significant,
and all Ps were two-sided.
% of total patients
(n ⫽ 111)
Mean % of cells staining
with antibody (⫾SD)
Mean antibody staining
intensity (⫾SD)
11
56
24
20
0.0 ⫾ 0.0
2.2 ⫾ 5.0
3.3 ⫾ 6.0
1.8 ⫾ 3.5
0.0 ⫾ 0.0
0.4 ⫾ 0.8
0.4 ⫾ 0.6
0.3 ⫾ 0.6
15
39
42
15
0.0 ⫾ 0.0
1.3 ⫾ 2.7
1.7 ⫾ 3.4
8.0 ⫾ 9.0
0.0 ⫾ 0.0
0.2 ⫾ 0.4
0.4 ⫾ 0.9
0.7 ⫾ 0.7
34
43
34
0.3 ⫾ 1.2
1.7 ⫾ 3.4
4.5 ⫾ 7.1
0.1 ⫾ 0.2
0.3 ⫾ 0.9
0.5 ⫾ 0.7
11
46
33
21
0.5 ⫾ 1.5
1.6 ⫾ 3.3
3.2 ⫾ 6.1
2.6 ⫾ 6.0
0.1 ⫾ 0.3
0.2 ⫾ 0.4
0.4 ⫾ 0.7
0.5 ⫾ 1.2
17
94
5.6 ⫾ 8
1.5 ⫾ 3.5
0.2 ⫾ 0.5
0.8 ⫾ 1.3
51
60
3.1 ⫾ 6.2
1.3 ⫾ 3.0
0.3 ⫾ 0.7
0.4 ⫾ 0.6
47
64
2.3 ⫾ 5.0
2.0 ⫾ 4.7
0.3 ⫾ 0.6
0.3 ⫾ 0.7
34
77
2.6 ⫾ 5.8
1.9 ⫾ 4.3
0.3 ⫾ 0.5
0.3 ⫾ 0.7
98
13
2.4 ⫾ 5.0
0.4 ⫾ 1.4
0.3 ⫾ 0.7
0.1 ⫾ 0.3
106
5
2.3 ⫾ 4.9
0.0 ⫾ 0.0
0.3 ⫾ 0.7
0.0 ⫾ 0.0
PIN, prostatic intraepithelial neoplasia.
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3066 Loss of 14-3-3␴ in Prostatic Tumorigenesis
normal prostate epithelium, expression of 14-3-3␴ was detected
in all samples (111 of 111; Table 2). The distribution of negative, weak, moderate, and strong staining for 14-3-3␴ was 0% (0
of 111), 3% (3 of 111), 35% (39 of 111), and 62% (69 of 111),
respectively. The median percentage of cells in each specimen
that stained positively for 14-3-3␴ expression was 86% in
normal epithelium.
The intensity and percentage of cells that reacted with
14-3-3␴ antibodies were significantly lower in PIN and invasive prostate adenocarcinoma (P ⬍ 0.001; Fig. 1; Table 2).
The majority of PIN cells (90%) showed negative or weak
14-3-3␴ immunoreactivity. Notably, none of the PIN cells
reacted strongly with 14-3-3␴ antibodies. An even more
dramatic decrease in 14-3-3␴ immunoreactivity was observed in invasive adenocarcinoma cells. In those specimens,
76% of adenocarcinoma cells lacked any 14-3-3␴ immunoreactivity. The little 14-3-3␴ immunoreactivity that was observed was primarily defined as weak staining (20%; 22 of
111) with some moderate staining in a few samples (4%; 4
of 111) and strong staining in 1% (1 of 111). The mean
percentages of the specimens with 14-3-3␴ staining in PIN
and invasive cancer were 4% (SD, 5.9) and 2% (SD, 4.8),
respectively.
In most cases, decreased 14-3-3␴ expression related to the
progression from normal epithelium to PIN. Ninety percent (100
of 111) of samples of PIN had either no (55%) or low (35%)
levels of 14-3-3␴ expression. The frequency and intensity of
14-3-3␴ expression further decreased from PIN (90% of negative or weak expression) to invasive cancer (97% of negative or
weak expression).
We also assessed whether other clinical parameters related
to 14-3-3␴ immunoreactivity. Adenocarcinomas with high
Gleason scores (⬎7) had significantly higher staining intensities
(0.5 ⫾ 0.7 versus 0.1 ⫾ 0.2; P ⫽ 0.008) and higher percentages
of 14-3-3␴ immunoreactive cells (4.5 ⫾ 7.1 versus 0.3 ⫾ 1.2;
P ⬍ 0.001) than adenocarcinomas with low Gleason scores (⬍7;
Fig. 2). Adenocarcinomas with lymph node metastases had
higher percentages of 14-3-3␴ expression (5.6 ⫾ 8 versus 1.5 ⫾
Table 2
3.5; P ⫽ 0.001), when compared with adenocarcinomas without
lymph node metastases. Overall, all invasive prostate adenocarcinomas had low intensities and low percentages (⬍5%) of
14-3-3␴ staining. There was no significant correlation between
the level of 14-3-3␴ expression and other clinical and pathological features, including patient age, extraprostatic extension,
vascular invasion, surgical margin, perineural invasion, or the
presence of high-grade PIN.
DISCUSSION
This is the first report that 14-3-3␴ expression is suppressed during the transformation of prostatic epithelium to
adenocarcinoma. Considering its function in the regulation of
the G2-M phase checkpoint and in the maintenance of
genomic integrity (6, 7), decreased 14-3-3␴ expression likely
contributes to increased mutability and well-established ability of malignant cells to overcome microenvironmental and
therapeutic challenges, which frequently characterize aggressive tumor cells.
In breast cancer, suppression of 14-3-3␴ arises as malignant behavior progresses from late atypical hyperplastic
lesions to ductal carcinoma in situ (12). Our data also indicate that decreased 14-3-3␴ expression similarly represents
an early event during the formation of prostate adenocarcinoma. Notably, 14-3-3␴ was not always uniform within a
particular specimen. These islands of prostate tumor cells,
which varied with regard to 14-3-3␴ expression, are likely
representative of the multifocal nature of prostatic adenocarcinoma (19). Alternatively, these differences may represent
tumor cell clones that have decreased 14-3-3␴ expression as
they progress toward a malignant phenotype. Nonetheless,
we should emphasize that our data cannot discriminate between a causal role for 14-3-3␴ in prostate cancer and the
alternative that the decreased 14-3-3␴ expression is an effect
of the carcinogenic process. We also note a paradoxical
increase of 14-3-3␴ expression as tumor progress (Fig. 2). In
some cases, islands of tumor cells with and without 14-3-3␴
Intensity (A) and percentage (B) of cells with 14-3-3␴ immunostaining in one hundred and eleven radical prostatectomy specimens
A
Staining intensity grade
Cell type
Normal
PINa,b
Adenocarcinomab,c
0
1
2
3
0 (0%)
61 (55%)
85 (76%)
3 (3%)
39 (35%)
22 (20%)
39 (35%)
11 (10%)
3 (3%)
69 (62%)
0 (0%)
1 (1%)
B
Cell type
Normal cells
PINd
Adenocarcinomad,e
Mean % of cells
staining ⫾ SD
Range (%)
86.3 ⫾ 7.8
4.1 ⫾ 5.9
2.2 ⫾ 4.8
40–100
0–25
0–25
a
PIN, prostatic intraepithelial neoplasia.
Indicates staining intensity was statistically lower compared to that of the normal cells with a P ⬍ 0.001 using a Cochran-Mantel-Henszel test.
c
The staining intensity was significantly lower compared to high-grade PIN (P ⫽ 0.002, Cochran-Mantel-Henszel test).
d
Indicates percentage of staining statistically lower compared with that of the normal cells with a P ⬍ 0.001 using a Wilcoxon-paired signed
rank test.
e
The percentage of staining was statistically lower compared to high-grade PIN (P ⫽ 0.001).
b
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Clinical Cancer Research 3067
Fig. 1 14-3-3␴ expression in the prostate. A-F, intraductal proliferation of neoplastic cells in high-grade prostatic intraepithelial neoplasia (PIN) are
often discontinuous and showed no or minimal immunoreactivity for 14-3-3␴. The adjacent normal epithelial cells showed strong immunostaining
for 14-3-3␴. G-L, in contrast to adjacent normal glands, tumor cells showed no or weak immunostaining for 14-3-3␴. ⽧, high-grade PIN. Arrows
indicate invasive carcinoma.
expression coexisted in the same specimens (Fig. 1L). This
suggested that clones of cells without 14-3-3␴ expression
may arise after cancer has developed. Cells with decreased
14-3-3␴ expression may develop mitotic failure as a result
of DNA damage. However, the cells that do survive have a
high probability of mutation and escape the growth restriction of their own mitotic clocks and surrounding microenvironments. This may explain the phenomenon that cancer
populations are not homogeneous (20, 21). For metastatic
cancer, single-modality treatment usually fails to eradicate cancer cells because resistant cells will arise during
treatment (22).
In conclusion, our findings are novel, in part, because
they show that 14-3-3␴ expression significantly decreases
during the progression from normal prostate tissue to PIN
and further to invasive cancer. We postulate that decreased
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3068 Loss of 14-3-3␴ in Prostatic Tumorigenesis
Fig. 2 Comparison of 14-3-3␴ expression in normal epithelium, highgrade prostatic intraepithelial neoplasia, and prostatic adenocarcinoma
(well-differentiated, GS ⬍ 7; moderately differentiated, GS ⫽ 7; and
poorly differentiated, GS ⬎7). There is a paradoxical increase of 143-3␴ expression as tumor dedifferentiated. GS, Gleason score.
14-3-3␴ expression contributes to the deregulation of genetic
stability, which frequently epitomizes invasive prostate
cancer.
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Loss of 14-3-3σ in Prostate Cancer and Its Precursors
Liang Cheng, Chong-Xian Pan, Jian-Ting Zhang, et al.
Clin Cancer Res 2004;10:3064-3068.
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