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Title Page
ADAM9 expression is decreasing in castration resistant prostate cancer and is a
prognostic factor for overall survival
All authors:
LIN Guo-wen*, YAO Xu-dong*, YE Ding-wei, ZHANG Shi-lin, DAI Bo, ZHANG Hai-liang
and MA Chun-guang
* These two authors contributed equally to this paper.
Department of Urology, Fudan University Shanghai Cancer Center, Shanghai 200032,
China(Lin GW, Yao XD, Ye DW, Zhang SL, Dai B, Zhang HL, Ma CG)
Department of Oncology, Fudan University Shanghai Medical College, Shanghai 200032,
China (Lin GW, Yao XD, Ye DW, Zhang SL, Dai B, Zhang HL, Ma CG)
Correspondence to: YE Ding-wei, Department of Urology, Fudan University Shanghai
Cancer Center, Shanghai 200032,China; Department of Oncology, Fudan University
Shanghai Medical College, Shanghai 200032, China (Tel: 86-21-64175590 ext.1807.
Fax: 86-21-64438640. E-Mail: [email protected])
This work was supported by Shanghai Cancer Center Foundation, the Natural Science
Foundation of China (No.30973009), and the Shanghai National Natural Science
Foundation (No.11ZR1407400).
Conflict of interest: none.
1
Abstract
Background A disintegrin and metalloprotease 9 (ADAM9) as one membrane-anchored
enzyme which is considered to be involved in some diseases including tumor, but it is still
not clear that the role of ADAM9 in castration resistant prostate cancer (CRPC). In this
study, we tried to explore the different expression on protein and messenger RNA (mRNA)
level of ADAM9 between hormonal sensitive prostate cancer (HSPC) and CRPC, and find
the correlation with prognosis.
Methods We collected and analyzed clinicopathologic characteristics of 106 HSPC and
76 CRPC cases, for which ADAM9 expression was compared by Immunohistochemistry.
ADAM9 mRNA of 32 additional cases(16 HSPC and 16 CRPC patients) were analysed via
quantitative real-time polymerase chain reaction (RT-PCR). The prediction values of
variables for overall survival (OS) of CRPC patients were analyze by Cox regression.
Results
ADAM9 protein expression was significantly downregulated in CRPC compared
with HSPC tissue (31.6% vs. 81.1%, P<0.001). The relativity transcription level of ADAM9
mRNA was 0.45 vs. 1.0 for CRPC and HSPC tissues, P=0.002. For CRPC group, patients
with ADAM9 protein low expression were significantly associated with shorter OS than
patients with high expression (38.6 months vs. 57.8 months, hazard rate [HR]=2.638,
P=0.023).
Conclusions
ADAM9 was low expression in CRPC correlated with poor prognosis and
might be involved in the succession from HSPC to CRPC by various functions. Hope to
have more large scale experiments to externally confirm our inference.
2
Key words:
ADAM9;
castration
resistant
prostate
cancer;
immunohistochemistry;
qRT-PCR;
prognostic factor
Although the quality of life has been improved and survival has been prolonged by the
hormonal therapy as the first treatment option for patients with advanced prostate cancer1,
2,
hormonal therapy is not a cure for prostate cancer and most patients are inevitable to
develop hormonal resistant disease with prostate specific antigen (PSA) relapse and
disease progression. Unfortunately, effective treatment options for castration resistant
prostate cancer (CRPC) are limited.3-6
Scholars try to explore the hormonal resistance mechanism and some hypotheses profile
part of prospect.7-9 In view of hormonal resistance is a complex process in which wide
variety of cytokines and signaling pathways are involved, the entire picture is still
ambiguous.
Cell-cell interaction mediated by various cytokines change or mutant in the
microenvironment always induce a lot of cascade reaction and play an important role of
3
affecting tumorigenesis, tumor cell survival, proliferation and invasion. In addition,
membrane-anchored enzymes such as matrix metalloproteinases and a disintegrin and
metalloproteinase (ADAM) family have critical action in the proteolytic processing of
various membrane bound molecules including growth factors, receptors and adhesion
molecules during the cell-cell interaction.10, 11 Therefore, ADAMs are considered to be
involved in some diseases including tumor.12, 13 In recent few years, ADAM9 was found to
play a significant role in tumorigenesis and progression where it was dysregulated.14-16
However, until now the effect of ADAM9 in CRPC is not clear. The present study aimed to
explore the expression of ADAM9 in CRPC, particularly focused on the relationship with
overall survival.
METHODS
Patients
A group of 182 patients with prostate adenocarcinoma were enrolled in the present study,
including 106 patients with hormonal sensitive prostate cancer (HSPC) and 76 cases of
CRPC from 2004 to 2011 at Shanghai Cancer Center of Fudan University in China . All
these patients’ characteristics were collected according to the clinical records and shown
in Table 1. Patients with CRPC were all in advanced stage with bone metastasis at
diagnosis and failure of first-line and second-line hormonal therapy with disease
progression and PSA increase. The definition of CRPC met the criteria of European
Association of Urology Guidelines.17 HSPC patients receive radical prostatectomy (n=50)
4
and androgen deprivation therapy (n=56) after diagnosis. All patients came to cancer
center for evaluation of disease and survival status every 4-6 weeks regularly.
Immunohistochemistry
182 prostate cancer tissues for these patients were obtained from tissue bank of Shanghai
Cancer Center. In addition, 30 tissues with benign prostate hyperplasia (BPH) as control
group were enrolled in this cohort. 50 HSPC tissues were from radical prostatectomy
specimens, whereas all CRPC and BPH tissues, and 56 cases of HSPC tissues were
obtained from transurethral prostatic resection. Tissue obtaining was conducted with
ethical approval of the local ethical committee.
Formalin-fixed paraffin-embedded tissue was freshly cut (4 mm), gradually hydrated
through xylene and ethanol. Antigen retrieval was achieved by pressure cooking at 97
centigrade in ethylene diamine tetraacetic acid (PH=8.0) for 10min. Slides were washing
thrice for 5 min in phosphate buffered saline and transferred to 3% H2O2 for 10 min to
block endogenous peroxidase activity. The primary ADAM9 antibody (primary rabbit
polyclonal antibody, AF949, R&D Systems, Wiesbaden of Germany18 was diluted 1:20 with
the use of a background reducing dilution buffer (Dako) and incubated overnight at room
temperature. Immunohistochemical analyses of ADAM9 were performed using labeled
streptavidin biotin method (LSAB kit, Dako) and diaminobenzidine(DAB, Dako) served as
chromogen. The slides were conventionally counterstained with haematoxylin and
mounted. ADAM9 positive prostate cancer cases omitting the primary antibody was
5
considered as negative control.
The immunostainings were evaluated independently by two pathologists, who unknew
patient pattern. The staining intensity(including cytoplasmic diffuse, the polar and luminally
accentuated form) was evaluated by semi-quantitative grading system, as negative, weak,
moderate or strong.18 The final results, however, were recorded as low (negative and weak
staining) or high (moderate and strong staining) expression.
Quantitative real-time polymerase chain reaction
Total RNA was prepared with TRIzol (Invitrogen, Cat#. 15596018) from manually
micro-dissected areas for 16 cases of HSPC and CRPC fresh tissue, respectively. DNase I
treatment and reverse transcription were performed with TaKaRa RNA PCR Kit (AMV)
according to the manufacturer’s instructions. Quantitative real-time polymerase chain
reaction (RT-PCR) was carried out and analyzed on a Mx3000P Quantitative PCR System
(Stratagene) using the ABsolute QPCR SYBR Green Mixes (ABgene, Cat#. AB-1166/b)
according to the standard protocol. The primer sequences of ADAM9 were
5’-CAGATGGCAAAAATCAAGCA-3’ and 5’-GATGGGAACTGCTGAGGTTG-3’. GAPDH
was
used
as
internal
control
with
the
primers
sequences
of
5’-TGACAACTTTGGTATCGTGGAAGG-3’ and 5’-GCAGGGATGATGTTCTGGAGAG-3’.
Statistical analysis
Data in this study included the following variables: age, clinical T, N and M stage, initial
6
PSA, biopsy Gleason score. In addition, for patients with CRPC, we collected nadir PSA
during former hormonal therapy, baseline PSA, alkaline phosphatase and hemoglobin at
the beginning of CRPC stage.
The Mann-Whitney U test and Χ 2 test was applied to identify the characteristics
difference between patients with CRPC and HSPC. The relationships between ADAM9
expression and patients’ characteristics were analyzed using Spearman’s rank correlations.
The univariate and multivariate Cox regression analysis were used to assess the
prognostic value of various variables to overall survival (OS) for patients with CRPC. The
median OS was computed using the Kaplan–Meier method. The differences in OS were
assessed by a log-rank test. The cut-off points of continuous variables including PSA at
diagnosis, nadir PSA and baseline PSA at CRPC were evaluated by the receiver operating
characteristics (ROC) and the Youden index, which is a good measure of diagnostic
effectiveness, calculated by sensitivity＋specificity－1.19,
20
When the maximal index
occurs at the cut-point, it will optimize the variable’s differentiating ability. P value of <0.05
was considered to be statistically significant difference. All statistical calculations were
computed by using SPSS version 13.0 (SPSS Inc, Chicago, IL).
RESULTS
Patients characteristics
The clinical and pathological variables for 182 prostate cancer patients were shown in
Table 1. There were no missing data for these variables except clinical T and N stage
7
because of the lack of records at diagnosis. Furthermore, only clinical M stage was
association with ADAM9 protein expression for HSPC patients (P<0.001, Table 2). The
median duration of follow-up was 48.3 months (range, 13–90 months). In CRPC group,
34.2% (26/76) patients had received chemotherapy based on docetaxel with average of
7.3 cycles. Other could not use the chemotherapy agent because of eld(17.1%),
inadequate organ functions(9.2%) and only used other chemotherapy strategy(39.5%). At
the end of follow-up, 73.3% (56/76) cases of CRPC died.
Decrease expression of ADAM9 protein in CRPC tissue
ADAM9 protein expression was found in CRPC, HSPC and BPH tissue. The membranous
and cytoplasmic was immunoreactivity staining as expected of a transmembrane protein
of ADAM9. Connective tissue, stromal tissue and vessels were negative for ADAM9
(Figure 1A, 1B, 1C). Compared to BPH tissue with ADMA9 high expression of 10.0%
(3/30), 81.1% (86/106) of HSPC cases had high expression of ADAM9, there were
significantly statistical difference between them, P<0.001. However, the percentage of
high expression dramatically decreased to 31.6% (24/76) for patients with CRPC, which
was significantly lower than HSPC tissue, P<0.001(Figure 1D).
Decrease transcription of ADAM9 mRNA in CRPC tissue
In quantitative RT-PCR test, we defined the relativity transcription level of ADAM9 mRNA
in HSPC tissue was one (standard error±0.122). Thus the relativity transcription level in
CRPC tissue was only 0.45 (standard error ± 0.106), 75% (12/16) tissue samples
8
transcription were less than the relativity value (Figure 1F). The outcome showed that
transcription of ADAM9 mRNA in CRPC tissue was significantly decreased compared to
that in HSPC tissue, P=0.002 (Figure 1E).
ADAM9 expression correlations with overall survival times
At the end of follow-up, the median OS for 76 patients with CRPC was 44.3 months (range,
21.6-86.0 months). Most variables entered into the survival analysis by univariate and
multivariate Cox regression method.
On the Cox regression analysis, ADAM9 protein expression, baseline hemoglobin at
CRPC and docetaxel chemotherapy were finally identified as the predictors of OS for
these patients (Table 3). The median PFS was 38.6 months for those with ADAM9 low
expression, compared with 57.8 months in ADAM9 high expression. The median PFS for
patients who had baseline hemoglobin of ≥120g/L and <120g/L were 56.3 months and
38.1 months, respectively. Patients with docetaxel chemotherapy had a median PFS of
49.9 months and the PFS in those without docetaxel chemotherapy decreased to 38.3
months. Additional analyses about the survival probability of ADAM9 protein expression
was further described in Figure 2.
DISCUSSION
ADAMs are membrane anchored glycoprotein family constructed by the protease and
adhesion domains multifunctional proteins, and contributed to the proteolytic processing of
9
other transmembrane proteins, cell adhesion and cell signaling events during the cell-cell
interaction and considered to be association with cancers.10, 11
ADAM9 is an important member of ADAM family and its dysregulated has been described
in various solid tumors and involved in the tumorigenesis. Most scholars reported that
ADAM9 had been ascribed important roles in invading their surrounding and metastasizing
to distant organs.21, 22 Thus, knockdown of ADAM9 resulted in reduced cellular migration
and invasion.14 Other functions of ADAM9, in fact, also contributed to cancer development
and progression. First, ADAM9 promoted pathological tumor vascularization.23 Second,
ADAM9
has
an
essential
role
in
the
transition
from
well-differentiated
to
poorly-differentiated carcinomas.24 Moreover, ADAM9 was a key factor in regulating
upstream and downstream cytokines such as epidermal growth factor receptor and
E-cadherin attached to various signaling pathways.25, 26
In the present study, we found ADAM9 higher expression in HSPC than BPH tissue, which
was similar to other reports.18 Furthermore, this study was designed to validate the
difference of ADAM9 expression in CRPC compared to the results for HSPC by
immunohistochemisty and quantitative RT-PCR. We noted that CRPC tissue displayed a
lower staining intensity in cancer cells than HSPC tissue (low expression rate: 68.4% vs.
18.9%,
P<0.001). The
quantitative
RT-PCR experiment
further confirmed this
phenomenon and also showed that ADAM9 mRNA reduced in transcription level in CRPC
tissue.
10
The relationship between the ADAM9 expression pattern in CRPC and the survival
probability was tested in survival analysis. By using a multivariate Cox proportional
hazards regression, ADAM9 expression was certified as an independent prognostic factor
of survival for CRPC patient. The lower expression of ADAM9, the worse OS for CRPC
patients were. Baseline hemoglobin and docetaxel chemotherapy presence were
considered as other two predictors of OS in the study, which was also confirmed by former
literatures.27, 28
The focus was that when prostate cancer transited from hormonal sensitiveness to
castration resistance, why the expression of ADAM9 was down regulation. As is known to
all, one of the main actions of ADAM9 in tumorigenesis is involved in metastasis.21, 22, 29
We observed that in the present study, all patients with CPRC had existence of metastasis
but only 31.6% (24/76) of cases was high expression of ADAM9. The expression
heterogeneity seemed to reveal that ADAM9 might play other crucial roles in the
succession from HSPC to CRPC.
One possible explanation for the phenomenon was that ADAM9 was more prominent in
the role of information access and cell communication, instead of promoting metastasis.
As described above, previous research articles confirmed that ADAM9 also assumed the
role of some signaling pathway ligands or receptors.30 It meant that when ADAM9
expression reduced, related signaling pathways enabling the transmission of information
11
might be missing or disorders, so as to induce some inconvertible exacerbations such as
cell differentiation from well differentiate to poor differentiate.11 It ultimately accelerated the
disease progression.
CONCLUSIONS
CRPC is a complex and heterogeneous tumor and the entire procedure of mechanism is
still cryptic. Our study suggested that ADAM9 was low expression in CRPC correlated with
poor prognosis and might be involved in the succession from HSPC to CRPC by various
functions. Hope to have more large scale experiments to confirm our inference.
12
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14
Table 1.
Patients Characteristics
Variables
CRPC Group
(n=76)
Age at diagnosis(yr)
Median
67.0
Range
56-80
Initial PSA(ng/mL)
Median
40.0
Range
13.0-142.0
Clinical T stage(No.)
T2
42
T3
25
T4
0
TX
9
Clinical N stage(No.)
N0
57
N1
10
NX
9
Clinical M stage(No.)
M0
0
M1
76
Biopsy Gleason score(No.)
6
13
7
24
8
10
9
17
10
12
ADAM9 protein expression(No.)
Low
52
High
24
nadir PSA (ng/mL)
Median
1.7
Range
0-44.5
Baseline PSA at
CRPC(ng/mL)
Median
36.0
Range
0.9-360.0
Baseline alkaline phosphatase at
CRPC(IU/L)
Median
103.0
Range
53.0-612.0
hemoglobin at CRPC(g/L)
Median
115.0
Range
93.0-196.0
HSPC Group
(n=106)
P value
0.117*
70.0
56-89
73.9
3.0-489.5
0.100*
0.088†
60
34
7
5
0.124†
74
26
6
<0.001†
69
37
0.065†
6
33
26
27
14
<0.001†
20
86
/
/
/
/
/
/
/
/
15
* Mann-Whitney U test was used for continuous variables; † Χ2 test was for Categorical
variables
16
Table 2. ADAM9 protein expression patterns correlated with clinicopathological
variables of prostate cancer
CRPC (n=76)
ADAM9 low
expression
Age at diagnosis (yr)
median value
Initial PSA (ng/mL)
median value
Clinical T stage (No.)
T2
T3
T4
Clinical N stage (No.)
N0
N1
Clinical M stage (No.)
M0
M1
Biopsy Gleason score
(No.)
6
7
8
9
10
nadir PSA (ng/mL)
median value
Baseline PSA at CRPC
(ng/mL)
median value
Baseline alkaline
phosphatase at CRPC
(IU/L)
median value
Baseline hemoglobin at
CRPC (g/L)
median value
ADAM9 high
expression
HSPC (n=106)
P value
‡
ADAM9 low
expression
ADAM9 high
expression
0.639
69
67
67
42
68
0.500
61.4
82.1
0.533
12
9
0
30
16
0
0.085
15
5
0
45
29
7
0.410
19
2
38
8
0.910
15
5
59
21
/
0
24
0
52
0.038
17
3
52
34
0.083
0
8
4
10
2
13
16
6
7
10
0.918
2
5
5
6
2
4
28
21
21
12
0.413
0.2
1.7
0.608
1.5
5.8
0.965
53.1
18.7
0.392
18.7
11.2
0.384
129
103
0.855
96
96
0.824
115
115
‡
0.515
0.746
43
P value
0.121
100
102
‡ The relationships between ADAM9 expression and patients’ characteristics were
analyzed using Spearman’s rank correlations.
17
Table 3.
Cox proportional hazards analysis of various clinical and pathologic
characteristics on correlating with overall survival for patients with CRPC
Univariate analysis
Age at diagnosis (year)
Initial PSA (ng/mL)
Clinical T stage
Clinical N stage
Biopsy Gleason score
ADAM9 expression
Nadir PSA (ng/mL)
Baseline PSA at CRPC
(ng/mL)
Docetaxel
chmeotherapy
Baseline hemoglobin
at CRPC (g/L)
Baseline alkaline
phosphatase at CRPC
(IU/L)
Multivariate analysis
<67 vs ≥67
<40 vs ≥40
T2 vs T3+T4
N0 vs N1
<8 vs ≥8
low vs high
<0.2 vs ≥0.2
P value
0.348
0.223
0.858
0.355
0.855
0.006
0.609
HR(95% CI)
1.291(0.757-2.202)
0.712(0.413-1.229)
1.056(0.581-1.919)
0.681(0.302-1.537)
1.050(0.619-1.782)
2.497(1.308-4.765)
0.869(0.507-1.490)
P value
0.378
0.163
0.692
0.653
0.915
0.023
0.887
HR(95% CI)
1.457(0.631-3.366)
0.576(0.266-1.249)
0.823(0.314-2.158)
0.792(0.287-2.188)
0.956(0.416-2.196)
2.638(1.143-6.091)
0.950(0.473-1.911)
<45 vs ≥45
0.323
1.310(0.767-2.239)
0.326
1.388(0.721-2.670)
No vs Yes
0.002
2.457(1.399-4.329)
0.038
2.288(1.047-5.000)
<120 vs ≥120
0.016
2.013(1.140-3.555)
0.005
3.279(1.420-7.575)
<130 vs ≥130
0.367
1.288(0.744-2.229)
0.249
1.580(0.726-3.437)
Note: Clinical M stage was not added in this analysis, because all CRPC patients had
metastasis.
18
Figure 1. Immunohistochemisty and quantitative real-time polymerase chain reaction
(RT-PCR) on ADAM9 for benign prostate hyperplasia, hormonal sensitive prostate cancer
and castration resistant prostate cancer. (A), (B), (C) was enlarged 400 times. (A) benign
prostate hyperplasia; (B) hormonal sensitive prostate cancer; (C) castration resistant
prostate cancer; (D) different ADAM9 protein expression in three groups ; (E) histogram
result of RT-PCR; (F) scatter plot result of RT-PCR.
19
Figure 2. A comparison of overall survival curve between patients with ADAM9 high
expression and low expression. The median PFS was 57.8months in patients with ADAM9
high expression, compared to 38.6months in patients with ADAM9 low expression.
20