Download Calcium-Binding Proteins S100A8 and S100A9 as Novel Diagnostic

Imaging, Diagnosis, Prognosis
Calcium-Binding Proteins S100A8 and S100A9 as Novel Diagnostic
Markers in Human Prostate Cancer
Alexander Hermani,1 Jochen Hess,2 Barbara De Servi,1 Senad Medunjanin,1 Rainer Grobholz,3
Lutz Trojan,4 PeterAngel,2 and Doris Mayer1
Abstract
Purpose: S100 proteins comprise a family of calcium-modulated proteins that have recently been
associated with epithelial tumors. We examined the expression of two members of this family,
S100A8 and S100A9, together with the S100 receptor RAGE (receptor for advanced glycation
end products) in human prostate adenocarcinomas and in prostatic intraepithelial neoplasia.
Experimental Design:Tissue specimens of 75 patients with organ-confined prostate cancer of
different grades were analyzed by immunohistochemistry for expression of S100A8, S100A9, and
RAGE. In addition, in situ hybridization of S100A8 and S100A9 was done for 20 cases. An ELISA
was applied to determine serum concentrations of S100A9 in cancer patients compared with
healthy controls or to patients with benign prostatic hyperplasia (BPH).
Results: S100A8, S100A9, and RAGE were up-regulated in prostatic intraepithelial neoplasia and
preferentially in high-grade adenocarcinomas, whereas benign tissue was negative or showed
weak expression of the proteins. There was a high degree of overlap of S100A8 and S100A9
expression patterns and of S100A8 or S100A9 and RAGE, respectively. Frequently, a gradient
within the tumor tissue with an increased expression toward the invaded stroma of the prostate
was observed. S100A9 serum levels were significantly elevated in cancer patients compared with
BPH patients or healthy individuals.
Conclusion: Our data suggest that enhanced expression of S100A8, S100A9, and RAGE is an
early event in prostate tumorigenesis and may contribute to development and progression or
extension of prostate carcinomas. Furthermore, S100A9 in serum may serve as useful marker to
discriminate between prostate cancer and BPH.
Prostate cancer is the most frequently diagnosed malignancy in
men and the second leading cause of cancer deaths among men
in Western countries (1). There is an urgent need for
appropriate diagnostic and prognostic markers, in addition to
the established serum protease prostate-specific antigen (PSA),
allowing an early diagnosis and the prediction of the clinical
behavior of individual tumors. Although the involvement of
certain genes and of chromosomal aberrations in prostate
carcinogenesis has been suggested (2, 3), the molecular
mechanisms underlying the initiation and progression of
prostate cancer are only poorly understood.
Authors’ Affiliations: 1Research Group Hormones and Signal Transduction and
2
Division of Signal Transduction and Growth Control, German Cancer Research
Center, Heidelberg, Germany; and Departments of 3Pathology, and 4Urology,
University Hospital Mannheim, University of Heidelberg, Mannheim, Germany
Received 2/15/05; revised 4/11/05; accepted 4/29/05.
Grant support: Tumorzentrum Heidelberg/Mannheim grant 781010.
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.
Note: Supplementary data for this article are available at Clinical Cancer Research
Online (http://clincancerres.aacrjournals.org/).
Requests for reprints: Doris Mayer, Research Group ‘‘Hormones and Signal
Transduction,’’ Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280,
69120 Heidelberg, Germany. Phone: 49-6221-423238; Fax: 49-6221-423237;
E-mail: d.mayer@ dkfz-heidelberg.de.
F 2005 American Association for Cancer Research.
Clin Cancer Res 2005;11(14) July 15, 2005
In an approach to identify genes that may play a role in
carcinogenesis (4, 5), we analyzed the expression of S100A8
and S100A9 in human prostate. The two proteins, S100A8
(calgranulin A) and S100A9 (calgranulin B), also designated as
MRP8 and MRP14, respectively, belong to the S100 multigenic
family of calcium-modulated proteins of the EF-hand type
(6, 7). Most of the 20 thus far identified S100 genes, including
S100A8 and S100A9, are located in a gene cluster on
chromosome 1q21, a region in which several rearrangements
that occur during tumor development have been observed (8).
Initially, S100A8 and S100A9 have been described mainly in
neutrophils and macrophages and were shown to be involved
in myeloid cell maturation (9, 10) and in inflammation
(reviewed in ref. 11). The proteins were suggested to form
S100A8/S100A9 heterocomplexes (12, 13), which were shown
to be secreted by activated monocytes (14). Expression of
S100A8 and S100A9 in epithelial tissues was first described in
context with squamous epithelia, e.g., various inflammatory
skin diseases (15), and with murine and human wound repair
(16). More recently, an association of S100 protein expression
with adenocarcinomas in humans has emerged. Immunohistochemical investigations have shown that S100A9 protein is
expressed in hepatocellular carcinomas, pulmonary adenocarcinomas, and invasive ductal carcinomas of the breast (17 – 19).
In these tumors, elevated expression of S100A9 was correlated
with poor differentiation. In patients with ovarian carcinomas,
S100A8 and S100A9 were found enriched in the cystic fluid and
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S100A8 and S100A9 in Prostate Cancer
in serum (20). Furthermore, overexpression of S100 mRNAs,
including S100A8 and S100A9, was reported for gastric cancers
(21). In contrast, S100A8 and S100A9 are frequently downregulated in poorly differentiated esophageal squamous cell
carcinomas (22, 23). Recently, expression of S100A2 and
S100A4, two other proteins of the S100 family, was shown to
be altered in human primary prostate cancer of different grades
(24). For S100A2, a progressive loss with increasing tumor
grade was observed, whereas S100A4 showed increased
expression in tumors with higher grades.
For several members of the S100 protein family, a function as
ligands for the receptor for advanced glycation end products
(RAGE) has been discussed (25, 26). RAGE is a cell surface
molecule that has been described as a multiligand receptor of
the immunoglobulin superfamily. The interacting ligands of
RAGE include advanced glycation end products, amphoterin,
h-amyloids, and S100 proteins (25, 27 – 29). Direct interaction
of S100 proteins with RAGE has been shown for S100A12 (ENRAGE), S100B, S100A1, and S100P (25, 26, 30), and it is
suggested that also other S100 family members are able to
modulate RAGE signaling. Engagement of RAGE by a ligand
triggers activation of central cellular pathways, including
mitogen-activated protein kinases, Cdc42/Rac, and nuclear
factor nB signaling pathways, thereby influencing features like
cell survival, cell motility, and inflammatory response (25, 31).
Blockade of RAGE signaling function was reported to lead to
decreased growth and metastasis of tumors in mice (31).
Expression of RAGE in prostate cancer was described with an
increased expression in metastatic compared with nonmetastatic cases (32).
In the present study, we investigated the expression of
S100A8 and S100A9 and of their putative receptor RAGE in
human prostatic tissue. In addition, we tested the value of
S100A9 as a serum marker for prostate cancer.
Materials and Methods
Tissue samples and patient sera. Prostate tissue was obtained with
consent from patients who underwent radical prostatectomy after
diagnosis of cancer. Tumors were diagnosed and classified according to
the Gleason system (33). H&E – stained paraffin sections serial to those
submitted to in situ studies were used for verification of the diagnosis in
the respective tissue specimens investigated. A total of 75 specimens
from 24 low-grade (Gleason score 5-6), 19 Gleason score 7, and 32
high-grade (Gleason score 8-10) organ-confined prostate cancers, scaled
according to Humphrey (34), were investigated. In these specimens, we
observed 18 prostatic intraepithelial neoplasias (PIN). In 48 specimens,
extended areas of benign prostatic tissue, including normal glands and
single hyperplastic glands, were present.
Serum samples were obtained before surgery with consent from
patients with subsequently diagnosed prostate cancer (n = 56) and
from patients with benign prostatic hyperplasia (BPH, n = 56) as well
as from 18 healthy men. Serum PSA of patients with BPH or cancer was
determined before surgery. At time of surgery, patients’ age ranged
between 55 and 82 years, and healthy men’s age ranged between 33
and 50 years.
Immunohistochemistry. For immunohistochemical staining of proteins, dewaxed formalin-fixed paraffin sections (4 Am) were rehydrated
and submitted to epitope retrieval by microwaving either in 0.1 mol/L
Tris (pH 9.5)/5% urea buffer for the detection of S100A8 and S100A9
proteins or in 0.01 mol/L citrate buffer (pH 6.0), respectively, for the
detection of RAGE. After blocking with 5% bovine serum albumin in
PBS, S100A8 and S100A9 were detected by specific rabbit polyclonal
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antibodies (Santa Cruz, Heidelberg, Germany, and kindly provided by
J. Roth, Institute of Experimental Dermatology, University of Münster,
Münster, Germany) and by the S100A9-specific mouse monoclonal
antibodies S36.48 (a generous gift of Dr. P. Pfeifer, BMA Biomedicals,
Augst, Switzerland) and 60B7 (kindly provided by R. Nozawa,
Laboratory of Host Defenses, University of Shizuoka, Shizuoka, Japan).
A rabbit polyclonal antibody (Santa Cruz) was used for detection of
RAGE. A peroxidase 3,3V-diaminobenzidine system (DAKO, Hamburg,
Germany) was used for the staining procedure. Finally, sections were
counterstained with hematoxylin and mounted in glycerol/gelatin.
For each antigen detected, staining intensity and area of positive
epithelial tissue were evaluated semiquantitatively. Staining intensities
were defined as moderate (1) or strong (2), whereas absent to faint
staining was considered as negative (0). For each section, areas with
similar staining intensity were evaluated together. The total areas with
moderate and strong staining, respectively, were estimated as percentage
of the total area of a given epithelial growth pattern observed in a
section. The cutoff for considerable positivity was set to 30%. When
both moderate and strong staining was detected in the same section, the
area representing the higher percentage of positivity, irrespective of the
staining intensity, was used for statistical evaluation. Exact permutation
test was used for comparison of benign prostatic tissue with tumor tissue
and Fisher’s exact test for comparison of low and high-grade tumors.
In situ hybridization. In situ hybridization of S100A8 and S100A9
mRNAs was done on 20 formaldehyde-fixed paraffin sections using
standard procedures. Sequences selected for the generation of riboprobes specific for S100A8 and S100A9 transcripts, as verified by
BlastN2 search, were amplified by reverse transcription-PCR using gene
specific primers for S100A8 (forward primer: 5V-ATTTCCATGCCGTCTACAGG-3V; reverse primer: 5V-TGGCTTTCTTCATGGCTTTT-3V) and
S100A9 (forward primer: 5V-CAGCTGGAACGCAACATAGA-3V; reverse
primer: 5V-CCACAGCCAAGACAGTTTGA-3V). PCR products representing nucleotides 128 to 329 (S100A8, Genbank accession no.
NM_002964) and nucleotides 64 to 537 (S100A9, Genbank accession
no. NM_002965) were cloned into pGEM-T vector (Promega,
Mannheim, Germany) and confirmed by sequencing. Digoxigeninlabeled antisense and sense riboprobes were generated by in vitro
transcription according to the digoxigenin application manual of
Roche (Mannheim, Germany). Hybridization was done at 55jC
overnight. Hybridized probes were detected using an alkaline
phosphatase – conjugated antidigoxigenin antibody (Roche) and an
alkaline phosphatase reaction using nitroblue tetrazolium/5-bromo-4chloro-3-indolyl phosphate as substrates.
ELISA. A sandwich immunosorbent assay for detection of S100A9
(BMA Biomedicals) was used to determine S100A9 concentrations in
serum. The immunoassay was carried out following the manufacturer’s
instructions. For statistical comparison of groups of individual values,
the Mann-Whitney test was applied and data were used for evaluation
of a receiver operator characteristic analysis.
Results
Immunohistochemical staining of human prostate tissue
sections from 75 cases of prostate cancer revealed extensive
expression of S100A8 and S100A9 proteins in adenocarcinomas of 58 (77%) and 51 (68%) cases, respectively. Protein
expression was evaluated semiquantitatively in tumor and
surrounding benign tissue. Both proteins showed a significant
up-regulation (P < 0.0001, S100A8 and P < 0.0001, S100A9)
in adenocarcinomas compared with benign prostatic tissue
(Table 1). Comparison of low-grade and high-grade cancers
revealed a preferential positivity of high-grade cancers (P =
0.053, S100A8 and P = 0.027, S100A9). Cancers diagnosed as
Gleason 7 showed an intermediate ratio of positive to total
number of cases compared with low-grade and high-grade
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Imaging, Diagnosis, Prognosis
Table 1. Evaluation of S100A8, S100A9, and RAGE positivity in human prostate cancer
Grading
n
c
Benign prostatic tissue
Low grade (Gleason score 5-6)
Gleason score 7
High grade (Gleason score 8-10)
PIN
48
24
19
32
18
S100A8
S100A9
RAGE
Positive cases
P*
Positive cases
P
Positive cases
P
7/48
15/24
13/19
28/32
8/18
<0.0001
5/48
14/24
9/19
28/32
8/18
<0.0001
2/48
12/24
13/19
23/32
6/18
<0.0001
0.053
0.053
0.027
0.016
0.16
0.013
NOTE: Positively stained tissue was evaluated. Low-grade cancers were compared with high-grade cancers and PIN with benign prostatic tissue using Fisher’s exact test.
*P < 0.05 was considered statistically significant.
cBenign prostatic tissue was compared with respective tumor tissue of the same cases using an exact permutation test. P values shown for benign prostatic tissue
document significantly lower expression of S100A8, S100A9, and RAGE in benign prostatic tissue than in adenocarcinomas of all grades.
cancers for S100A8. For S100A9, the relative amount of
positive Gleason 7 cases was lower than in low-grade cancers.
There was no obvious difference between cancer grades
concerning staining intensity.
A marked overlap between S100A8 and S100A9 staining
patterns was apparent in corresponding tissue areas of serial
sections (Fig. 1A and B). S100A8 and S100A9 protein
expression was detected in single benign prostate glands usually
adjacent to tumor tissue and commonly restricted to the basal
cell layer (Fig. 1C). Benign hyperplastic glands showed no, or
only weak, expression of the S100 proteins (Fig. 1D). In
addition, positive staining was found in 8 of 18 PINs,
indicating an up-regulation of S100A8 and S100A9 expression
also in precancerous lesions (Fig. 1E).
Fig. 1. Immunohistochemical staining of
S100A8 (A) and S100A9 (B-L) protein in
human prostate tissue. A and B, serial
sections of an adenocarcinoma stained with
S100A8 and S100A9 antibodies showing
an overlapping expression pattern;
C, benign gland showing S100A9-positive
basal cells; D, weak to absent staining of
S100A9 in hyperplastic glands; E, positive
staining of PIN; F, expression gradient with
increased positivity of tumor cells toward
the stroma; G, S100A9 expression in a
perineural tumor, nf, nerve fiber;
H, high-grade adenocarcinoma with
cribriform pattern; I, partially or completely
stained glands beside negative glands;
J, heterogeneous expression pattern of
S100A9 in a low-grade tumor; K , tumor
cells with nuclear staining; L, positive tissue
histiocytes demonstrating specificity of
antibody reaction. Magnifications: 45
(A and B), 120 (C, I, K , and L), and
60 (D-H and J).
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S100A8 and S100A9 in Prostate Cancer
In 27 of 75 cases (36%), a staining gradient for the two
S100 proteins was observed with an increased staining
intensity in tumor cells adjacent to the stromal compartment
or the capsule of the prostate (Fig. 1F). This gradient pattern
was observed in carcinomas independently from the tumor
grade. In addition, S100A8 and S100A9 expression was
frequently observed in tumor tissue surrounding nerve fibers
(Fig. 1G). In high-grade tumors, as shown for a cribriform
tumor gland (Fig. 1H), and also in other growth patterns, a
marked heterogeneity in immunoreactivity was generally
observed. Tumor glands showing no staining were found
adjacent to partly positive and strongly positive glands (Fig.
1I). Typically, groups of tumor glands or individual glands as
well as single cells within the glands showed positive reaction
(Fig. 1J). In 29 of 75 cases (39%), nuclear staining in addition
to, or instead of, cytoplasmic staining was observed at least in
subpopulations of cells (Fig. 1K), although without visible
correlation with the tumor grade. The different antibodies used
for detection of S100 proteins yielded similar staining patterns.
S100A8 and S100A9 were detected in tissue histiocytes with all
antibodies tested (Fig. 1L) independent of the histopathologic
properties of different specimens, indicating a specific reaction
under the experimental conditions applied. Additional staining was found in blood vessels, whereas stromal cells usually
showed no specific staining reaction, although positively
stained areas where observed in the stromal compartment.
Expression of S100A8 and S100A9 by stromal cells, however,
was not detected by RNA in situ hybridization of S100A8 and
S100A9 mRNAs on prostate tissue sections using specific
probes.
In situ hybridization revealed a marked overlap in S100A8
and S100A9 mRNA expression patterns in agreement with
results obtained by immunohistochemistry. Positive reaction
was observed in basal cells of benign glands (Fig. 2A and D)
and in adenocarcinomas of different grades (Fig. 2B, E, G, and
H). The staining showed varying intensity in different positive
tumor areas, including PIN, the latter showing positive
staining both in basal cells and in intraepithelial neoplastic
cells (Fig. 2I).
Expression of the S100 receptor RAGE in prostate cancer was
previously reported by Kuniyasu et al. (32). We were interested
whether expression of RAGE coincides with the expression of
S100A8 and S100A9. Immunohistochemical analysis of RAGE
on the same set of prostate cancer tissues used for detection of
the S100 proteins revealed a marked overlap of RAGE with
S100A8 or S100A9 staining patterns (Fig. 3A-D and Supplemental Figs. S1 and S2). Also, for RAGE, a strong expression in
tumor tissue invading the stroma or adjacent to connective
tissue of the prostate capsule was observed (Fig. 3E). In
addition, similar to S100A8 and S100A9, strong positivity was
found in perineural tumor tissue (Fig. 3F). RAGE expression
was detected in adenocarcinomas of different grades with a
weak trend to increased positivity of high-grade cancers (P =
0.16; Table 1).
We next addressed the question of a putative diagnostic
value of the S100 proteins as serum markers. For this purpose,
an ELISA that was specific for S100A9 was selected.
Comparing S100A9 serum levels of 37 cases of prostate
cancer with 18 healthy controls, we discovered increased
concentrations of S100A9 in serum of cancer patients (P =
0.0037; Fig. 4A). This observation, together with the finding of
poor expression of S100A9 in benign hyperplastic tissue,
prompted us to compare a larger number of prostate cancer
cases (n = 56) with individuals diagnosed for BPH (n = 56).
Fig. 2. Analysis of S100A8 and S100A9
mRNA expression by in situ hybridization.
A and D, normal glands showing expression
of S100A8 (A) and S100A9 (D) in basal
cells; B and E, serial sections of an
adenocarcinoma hybridized with S100A8
(B) and S100A9 (E) antisense probes
showing an overlapping expression pattern
of S100A8 and S100A9; C and F,
hybridization results with the respective
labeled S100A8 (C) and S100A9 (F) sense
probes as negative control; G to I, in situ
hybridization of S100A9, tumor with an
adjacent benign gland (G, arrow),
high-grade adenocarcinoma (H), and
PIN (I). S100A9 is expressed in basal cells
and in intraepithelial neoplastic cells.
Magnifications: 120 (A, D, and G) and
60 (B, C, E, F, H, and I).
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Imaging, Diagnosis, Prognosis
Fig. 3. Detection of RAGE by immunohistochemistry. A to C, serial sections of prostate cancer tissue stained for RAGE (A), S100A8 (B), and S100A9 (C) showing an
overlapping expression pattern; D, expression pattern of RAGE in a section serial to Fig. 1A and B, which show similar patterns of S100A8 and S100A9 staining; E, increased
RAGE staining intensity in tumor tissue adjacent to stroma; F, RAGE-positive perineural tumor; Magnifications: 45 (A-E); 60 (F).
We found significantly increased S100A9 serum levels in
prostate cancer patients in comparison with BPH patients (P <
0.0001; Fig. 4B), the latter exhibiting values that were similar
to values obtained for healthy individuals. These data indicate
that elevated S100A9 serum concentrations are connected with
cancer patients but not with patients suffering from a benign
prostatic disease. S100A9 levels of low-grade and high-grade
cancer patients were both elevated without significant differences
Fig. 4. S100A9 serum concentrations in
prostate cancer patients (CaP) compared
with healthy controls and BPH. A, box plot
showing elevated S100A9 serum levels in
patients with prostate cancer compared
with healthy men (*P = 0.0037);
B, S100A9 levels are significantly increased
in cancer patients compared with BPH
patients (**P = 1.6 10 7); C and D,
receiver operator characteristic analysis of
S100A9 and PSA comparing BPH and
prostate cancer for all patients analyzed (C)
and for patients with PSA < 10 ng/mL (D).
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S100A8 and S100A9 in Prostate Cancer
between the two groups. A comparison of S100A9 concentrations and the corresponding PSA serum levels in a receiver
operator characteristic analysis revealed that S100A9 values
provided a diagnostic reliability that was similar to the one
based on PSA values in the cases analyzed (Fig. 4C). However,
PSA and S100A9 were independent parameters. The evaluation
of S100A9 serum levels of patients with PSA values <10 ng/mL
still allowed a highly significant discrimination of BPH and
cancer by S100A9 (P < 0.0001), exceeding that achieved with
PSA (P = 0.02; Fig. 4D; Table 2).
The present data show that S100A8 and S100A9 mRNA and
proteins are up-regulated in PIN and in prostatic adenocarcinomas of different histopathologic grades. The expression
patterns of both proteins are similar to that observed for the
S100 receptor RAGE. S100A9 was additionally detected with
significantly increased levels in sera of prostate cancer patients,
whereas individuals with BPH displayed S100A9 levels within a
reference range.
Discussion
S100 proteins are involved in a variety of biological
processes and are considered to be expressed in a cell type –
or tissue-specific manner. However, deregulated expression of
several members of the S100 family, including S100A8 and
S100A9, under various pathologic conditions has been
reported (7, 35). Besides the well-documented expression in
inflammatory skin diseases (15), S100A8 and S100A9 and
other members of the S100 family recently have been
associated with rheumatoid arthritis (36), psoriasis (37),
cutaneous wound repair (16), and various experimental and
human neoplasms (4, 17 – 19). It is noteworthy that the
patterns of differential gene expression in cancer, particularly
in prostate and liver cancer, strongly correlate with the pattern
of differential gene expression during wound-healing processes (38). These observations suggest additional functions of the
S100 protein family in injury and disease pathogenesis. Our
findings on the expression of S100A8 and S100A9 in prostatic
adenocarcinomas support this hypothesis. Using immunohistochemistry and RNA in situ hybridization on human prostate
samples of patients with diagnosed prostate cancer, we could
show that S100A8 and S100A9 are up-regulated in prostatic
Table 2. Statistical evaluation of S100A9 and PSA
serum levels for dissection of BPH patients from
cancer patients
Patient group
n
CaP
PSA > 0 ng/mL
PSACaP < 10 ng/mL
PSACaP/BPH < 10 ng/mL
56
34
34
P
BPH
56
56
49
S100A9
1.6 10
5.1 10
1.3 10
7
6
5
PSA
1.5 10
0.02
0.0006
6
NOTE: P values were calculated, using Mann-Whitney test, for comparison of
S100A9 and PSA in cancer patients with BPH patients grouped regarding
PSA levels.
Abbreviation: CaP, prostate cancer.
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adenocarcinomas and in PIN, suggesting an early involvement
of the proteins in prostate cancer. It is intriguing that the
tumor cells, as precursors of which the luminal cells are
considered (39), greatly restore the ability of S100A8 and
S100A9 expression, whereas in benign glands expression was
found only in the basal cell layer, which is responsible for the
epithelial/stromal contact. Recently, different compounds that
also participate in the formation of extracellular matrix of
connective tissue or basement lamina were found to associate
with S100 proteins. For neutrophils, involvement of S100A8
and S100A9 in the modulation of transendothelial migration
by interaction with glycosaminoglycans and carboxylated
glycans has been shown (40, 41). In addition, S100A8 and
S100A9 have been described to increase adhesion of monocytes to fibronectin (42). Worth mentioning in this context is
the observed close spatial correlation of S100A8- and S100A9positive basal cells and the stroma on the one hand and
positive cancer cells and stroma on the other hand (see also
Supplemental Fig. S2). Our finding of increased S100A8,
S100A9, and RAGE expression toward the invaded connective
tissue further suggests a functional relation with the extension
of epithelial tissue. With respect to migration events, an
involvement of RAGE engaged by S100B, S100A1, and
amphoterin has been associated with neurite outgrowth
(26, 28). Furthermore, RAGE has been shown to play an important role for tumor growth and metastases formation in
mice (31). Engagement of RAGE is known to trigger different
signaling cascades, usually leading to activation of the transcription factor nuclear factor nB (25, 26, 43). Interestingly,
overexpression of p65, the active subunit of nuclear factor nB,
was found to occur as an early event in the development of
prostate cancer (44). Furthermore, active nuclear factor nB was
frequently detected in prostatic adenocarcinomas with perineural location and has been linked functionally to mechanisms that promote perineural invasion (45), which is the
major mechanism of prostate cancer spread outside the
prostate (46). Together with our findings of enhanced
expression of RAGE and S100A8/S100A9 in prostate cancer
and of frequent positivity of perineural tumor tissue (see also
Supplemental Figs. S1 and S2), it is intriguing to speculate
that S100/RAGE signaling events may be involved in the
development of survival or growth advantages via nuclear
factor nB. Therefore, the S100/RAGE signaling pathway may
represent a suitable target for prevention or treatment
strategies for prostate cancer. In addition, our data provide
evidence for a diagnostic value of the S100 proteins. We
found a significant increase of S100A9 serum concentrations
in prostate cancer patients compared with healthy individuals
or to BPH patients. In comparison to PSA, currently the best
diagnostic serum marker for prostate cancer, S100A9 distinguished with a higher sensitivity between BPH and cancer
patient groups with low PSA levels. Thus, S100A9 may
represent a helpful candidate for the dissection of prostate
cancer and BPH cases, where PSA measurement fails to
provide reliable diagnostic information. Most recently,
S100A9 was discovered by another group in voided urine
after prostatic massage from patients with prostate cancer
using two-dimensional gel electrophoresis (47). Together with
the present study, this finding further supports the putative
diagnostic value of S100A9 measurement in body fluids as a
marker for prostate cancer.
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In summary, our data show that S100A8 and S100A9 are
up-regulated in adenocarcinomas of the prostate and in
PIN. The expression of both proteins markedly overlaps
with the expression of the S100 receptor RAGE, which has
been associated with invasive potential of different tumors.
The use of S100A9 as a serum marker for the early
detection of prostate cancer additionally may help to
facilitate dissection of prostate cancer patients from patients
with BPH.
Acknowledgments
We thank A. Benner (Central Unit of Biostatistics, German Cancer Research Center, Heidelberg, Germany) for advice with the statistical analysis and C. Gebhardt for
helpful discussion; J. Roth (Institute of Experimental Dermatology, University of
Mu«nster, Germany)for generously providing the rabbit polyclonal antibodies against
S100A8 and S100A9; P. Pfeifer (BMA Biomedicals AG, Augst, Switzerland) for the
gift of monoclonal antibody clone S36.48; and R. Nozawa (Laboratory of Host
Defenses, University of Shizuoka, Japan) for generously providing the monoclonal
antibody 60B7.
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Calcium-Binding Proteins S100A8 and S100A9 as Novel
Diagnostic Markers in Human Prostate Cancer
Alexander Hermani, Jochen Hess, Barbara De Servi, et al.
Clin Cancer Res 2005;11:5146-5152.
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