Prostate Cancer

ADAM9 Expression is a Significant and IndependentPrognostic Marker of PSA Relapse in Prostate Cancer

e u r o p e a n u r o l o g y 5 4 ( 2 0 0 8 ) 1 0 9 7 – 1 1 0 8

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Florian R. Fritzsche a, Monika Jung b, Angelika Tolle b, Peter Wild c, Arndt Hartmann d,Kirsten Wassermann a, Anja Rabien b, Michael Lein b, Manfred Dietel a, Christian Pilarsky e,Daniela Calvano e, Robert Grutzmann e, Klaus Jung b, Glen Kristiansen a,c,*a Institute of Pathology, Charite - Universitatsmedizin Berlin, Berlin, GermanybDepartment of Urology, Charite - Universitatsmedizin Berlin, Berlin, Germanyc Institute of Pathology, Universitatsspital Zurich, Zurich, Switzerlandd Institute of Pathology, University of Regensburg, Regensburg, GermanyeDepartment of Surgery, University Hospital Dresden, Dresden, Germany

Article info

Article history:Accepted November 12, 2007Published online ahead ofprint on November 26, 2007

Keywords:ADAM9ImmunohistochemistryqRT-PCRPrognostic markerProstate cancer

Abstract

Objectives: A disintegrin and metalloprotease (ADAM) 9 has been impli-cated in tumour progression of prostate cancer. We evaluated theexpression of ADAM9 on protein and messenger RNA (mRNA) level ina larger cohort of prostate cancer cases following prostatectomy andcorrelated the findings with clinicopathological parameters includingprostate-specific antigen (PSA) relapse times.Methods: We immunostained 198 clinicopathologically characterisedprostate cancer cases for ADAM9. For 25 additional cases, ADAM9 mRNAof microdissected tumour and normal tissue was analysed via quanti-tative reverse transcriptase-polymerase chain reaction.Results: ADAM9 was significantly upregulated in prostate cancer com-pared with normal tissue on mRNA and protein level. ADAM9 proteinexpression was significantly associated with shortened PSA relapse-freesurvival in univariate and multivariate analyses, particularly in patientswho had received prior androgen ablation.Conclusions: ADAM9 is overexpressed in prostate cancer cases and is an

gnostic marker of PSA relapse-free survival followingtomy. Further studies are needed to verify its role as a

independent proradical prostatec

r of response to androgen ablation.ociation of Urology. Published by Elsevier B.V. All rights reserved.

predictive marke# 2007 European Ass

* Corresponding author. Institute of Surgical Pathology, University Hospital Zurich,Schmelzbergstr. 12, CH-8091 Zurich. Tel. +41 44 2553457; Fax: +41 44 2554416.E-mail address: glen.kristiansen@usz.ch (G. Kristiansen).

0302-2838/$ – see back matter # 2007 European Association of Urology. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.eururo.2007.11.034

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1. Introduction

In this study we focused on ADAM9, a member of the‘‘A disintegrin and metalloprotease’’ (ADAM) family,in prostate cancer. This family of proteins isinvolved in different physiological and pathophy-siological functions including cell adhesion, cellmigration, and tissue remodeling [1]. ADAMs aremembrane-anchored glycoproteins that consist of aprotease and an adhesion domain. The interactionsof ADAMs with cell-surface and extracellular matrixproteins could be of relevance to tumour biologybecause these processes are vital for tumourprogression [2–4]. Concordantly, various ADAMshave been shown to represent a potential diagnosticand prognostic marker in human tumours [5].

The type I transmembrane protein ADAM9(synonyms, MDC9, meltrin-g) is a cell adhesionmolecule, found to interact with avb5 integrin, tocleave the insulin B-chain, and to be involved in theectodomain shedding of membrane-anchoredheparin-binding epidermal growth factor (EGF)-likegrowth factor [4,6,7]. In addition, a secreted form ofADAM9 that resulted from alternative splicing wasdescribed [8]. ADAM9 might be essentially involvedin carcinogenesis and tumour progression by med-iating EGF receptor activity and by promoting cancer

Table 1 – Associations (chi-square tests) between the proteinclinicopathological parameters

Total

All cases 198

Age

�62 105

>62 93

Pre-OP PSAb

�9.4 ng/ml 87

>9.4 ng/ml 82

PT status

pT2 110

pT3/4c 88

Gleason sumd

3–6 89

7 56

8–10 52

Residual tumoure

R0 109

R1 87

ADAM9, A disintegrin and metalloprotease 9; Pre-Op PSA, preoperative Pa Dichotomisation was achieved according to the median (100).b Preoperative PSA was not available for 29 cases.c Five cases were pT4.d For one case no Gleason score was given in the report.e Two cases were Rx.

cell invasion via regulation of E-cadherin andintegrins [6,9–15].

In this study we evaluated ADAM9 expression onprotein and transcript level to clarify a diagnostic orprognostic value of ADAM9 in human prostatecancer.

2. Methods

2.1. Patients (reverse transcriptase-polymerase chainreaction)

Twenty-five matched (tumour/normal) prostate samples were

microdissected from frozen sections. Cases used for micro-

dissection and messenger RNA (mRNA) preparation differed

from the cohort used for immunohistochemistry.

2.2. Patients (immunohistochemistry)

One hundred ninety-eight prostatectomy patients were

enclosed in this study. Patient age ranged between 47 and

74 yr (median, 62). Preoperative prostate-specific antigen (PSA)

levels ranged from 0.03 to 150 ng/ml (median, 9.4). Forty-four

patients (22.2%) had received gonadotropin-releasing hor-

mone analogues at the discretion of the referring urologist

prior to surgery (median, 4 wk; range, 2–16 wk). Because this

short-term antiandrogenic therapy did not result in morpho-

logically discernable effects, Gleason score was evaluated as

expression of ADAM9 in prostate cancer and

Low ADAM9a High ADAM9a p value

85 (42.9%) 113 (57.1%)

0.010

36 (34.3%) 69 (65.7%)

49 (52.7%) 44 (47.3%)

0.877

35 (40.2%) 52 (59.8%)

34 (41.5%) 48 (58.5%)

0.471

50 (45.5%) 60 (54.5%)

35 (39.8%) 53 (60.2%)

0.597

40 (44.9%) 49 (55.1%)

24 (42.9%) 32 (57.1%)

21 (40.4%) 31 (59.6%)

0.663

48 (44.0%) 61 (56.0%)

35 (40.2%) 52 (59.8%)

SA; pT status, tumour stage.

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usual. Clinical follow-up data, as annually assessed, including

PSA relapse-free survival time were available for all patients.

The median follow-up time of all cases was 46.5 mo (range,

1–180). The median follow-up time of patients without a PSA

relapse was 48 mo (range, 10–180). Sixty-nine patients (34.8%)

experienced a PSA relapse after a median time of 31 mo (range,

3–139). The Gleason scores (GS) in the cohort were distributed

as follows: GS 2–6, 89 (44.9%); GS 7, 56 (28.3%); GS 8–10, 52

(26.3%); missing data, 1 (0.5%) (Table 1).

2.3. Quantitation of ADAM9 mRNA

The total RNA isolation was performed with the use of RNeasy

Micro Kit (Qiagen, Hilden, Germany) from manually micro-

dissected areas of normal prostate tissue, prostatic intrae-

pithelial neoplasia (PIN), and prostate cancer, specified by a

genitourinary pathologist and previously described in detail

[15].

RNA concentrations were measured with the NanoDrop

ND-1000 Spectrophotometer (NanoDrop Technologies, Wil-

mington, DE, USA). The integrity of isolated RNA was assessed

with the use of the RNA 6000 Nano LabChip1 kit and the

Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA,

USA). All samples were characterised by an A260/A280 ratio

>1.90 and an RNA integrity value >7.

Complementary DNA (cDNA) synthesis was performed

with the use of the Transcriptor First Strand cDNA Synthesis

Kit (Roche Applied Science, Penzberg, Germany) and a

maximum of 1 mg RNA in reaction. The cDNA volume

amounted to 20 ml.

Real-time polymerase chain reaction (PCR) was performed

with the LightCycler Instrument (Roche Applied Science).

Prediluted (1:5) cDNA was used for the quantification of

reference gene and target gene expression. The PCR reaction

volume was 10 ml including 1 ml diluted cDNA. The PCR run

conditions of the three reference gene quantifications were

the same as described previously [16]. Paired samples of

nonmalignant and malignant tissue areas were measured in

one PCR run. The reaction condition for ADAM9 mRNA and for

its two transcript variants were established separately for

each PCR. The real-time detection of PCR products was

performed with fluorescence dye SYBR green I (QuantiTect

SYBR green PCR Kit; Qiagen); reaction conditions are given in

Table 2. Only one single melting peak was observed for PCR-

specific amplicons. For all genes, calibration curves with

Table 2 – Real-time reverse transcriptase-PCR data with prime

Target gene and transcript variants ACC no.

Total ADAM 9 NM_003816

Transcript variant 1 (transmembrane) NM_003816

Transcript variant 2 (cytosolic) NM_001005845

PCR, polymerase chain reaction; ACC no, GenBank accession number; P

disintegrin and metalloprotease 9.

pooled cDNA were generated. PCR efficiencies were amounted

from cDNA dilution curves of pooled cDNA. Each PCR run

included a cDNA with known expression level and was used as

a standard for quantification of an unknown sample calcu-

lated by LightCycler Software, version 3.5 (Roche Applied

Science). For relative quantification of ADAM9 mRNA and both

transcript variants, the expressions were normalised on

geometric mean of the expression of the three reference

genes hypoxanthine-phosphoribosyl-transferase (HPRT1),

delta aminolevulinate synthase 1 (ALAS1), and K-alpha-1

tubulin (K-ALPHA-1) [16].

2.4. Western blot analysis

Paired tissue samples (cancer/normal) from six prostatect-

omy specimens were used for protein extraction. After

histological control to ascertain the specific histology the

tissue was frozen, minced, and homogenised by an ultra-

sound device in 50 mmol/l Tris-HCl, 10 mmol/l CaCl2, pH 7.5,

with 0.25v/v% Triton1 X-100 (Sigma-Aldrich Chemie GmbH,

Munich, Germany), in the presence of protease inhibitors

(0.1 mmol/l phenylmethylsulphonyl fluoride, 1 mg/ml apro-

tonin, 10 mg/ml soybean trypsin inhibitor). After centrifuga-

tion at 23,000g for 15 min at 4 8C, the supernatants were stored

at �80 8C until analysis. Twenty micrograms of total protein

of each tissue sample were applied for fractionation on an

sodium dodecyl sulphate-polyacrylamide gel electrophoresis

(10% w/v polyacrylamide, reducing conditions) and then

transferred onto a polyvinylidene difluoride membrane

(Millipore Corp, Bedford, MA, USA). The membrane was

incubated with antihuman ADAM9 polyclonal antibody

(0.2 mg/ml; CEDARLANE LABORATORIES Limited, Ontario,

Canada). Actin (anti–b-actin clone AC-74; Sigma-Aldrich

Chemie GmbH) served as a loading control. Horseradish

peroxidase–conjugated goat antirabbit immunoglobulin G

(IgG) and rabbit antimouse IgG (Dako, Hamburg, Germany)

were used as secondary antibodies. The antigen-antibody

reaction was visualised by enhanced chemiluminescence

(Amersham Biosciences, Piscataway, NJ, USA). Intensity of

the detected signals by Western blot was quantified with the

use of Fluor-S MultiImager (Bio-Rad Laboratories, Hercules,

CA, USA).

Specificity of ADAM9 bands were confirmed with the use of

(a) blocking peptide, corresponding to carboxy terminal end of

human ADAM9, at 30-fold concentration excess of the primary

r sequences

Primer sequence 50. . .. . .30 PS (bp) E

F: cag atg gca aaa atc aag ca 151 2.01

R: gat ggg aac tgc tga ggt tg

F: gga tac gga gga agt gtg ga 110 1.91

R: gca cag aca ata agg gga aca

F: gtc atg gac atg gga aat ga 120 1.95

R: ctc cac agt tga tcc ctc ttg

S, PCR product sizes; E, efficiencies of target gene PCRs; ADAM9, A

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antibody, and (b) control IgG of serum from healthy non-

immunised rabbits (Dako).

2.5. Immunohistochemistry

Formalin-fixed paraffin-embedded tissue was freshly cut

(3 mm), mounted on superfrost slides (Menzel Glaser),

dewaxed with xylene, and gradually hydrated. Antigen

retrieval was achieved by pressure cooking in 0.01 mmol/l

citrate buffer for 5 min. The primary ADAM9 antibody

(primary goat polyclonal antibody, AF949; R&D Systems,

Wiesbaden, Germany) [17] was diluted 1:50 with the use of a

background reducing dilution buffer (Dako) and incubated at

room temperature for 1 h. This antibody (AF949) is specific for

mouse ADAM9 and cross-reacts with human ADAM9. It does

not cross-react with human ADAM8, ADAM10, ADAM15, or

ADAM17/TACE (data not shown). Detection took place by the

conventional labelled-streptavidin-biotin method (LSAB-kit,

Dako) with Fast-Red (Sigma-Aldrich Chemie GmbH) as

chromogen. The slides were counterstained and mounted.

Antibody specificity was confirmed by a blocking experiment,

coincubating the antibody with the corresponding ADAM9

peptide (949-AD, R&D Systems), which abolished immuno-

reactivity. As negative controls we used ADAM9-positive

prostate cancer cases omitting the primary antibody.

2.6. Evaluation of the immunohistochemical stainings

The immunostainings were evaluated by two genitourinary

pathologists. To assess staining intensity and quantity of cells

stained, we applied the H-score separately for prostate cancer,

adjacent normal prostatic tissue (peripheral zone), and PIN.

The staining intensity (including cytoplasmic diffuse and the

polar, luminally accentuated form) was evaluated with a four-

tier grading system (0 = negative, 1 = weak, 2 = moderate, and

3 = strong staining intensity). For each staining intensity

grade, the percentage of tissue stained was multiplied by

the corresponding intensity grade (0–3). Theses values were

summed for each tissue type, resulting in a score between 0

and 300. To delineate between low and high levels of ADAM9

expression, the median H-score was used to dichotomise the

data.

2.7. Statistical analysis

Statistical analysis was performed with SPSS, version 14.0

(SPSS Inc, Chicago, IL, USA). p values < 0.05 were considered

significant.

Fig. 1 – ADAM9 messenger RNA (mRNA) expression. (A)

Normalised mRNA expression values of ADAM9. The

columns are means W standard error of the mean.

Significances calculated by paired t tests. (B) Multiple of

normalised prostate cancer (PC) ADAM9 expression to

adjacent normalised prostate tissue (PN) divided according

to the splicing variants (see A). For all splicing variants the

majority of prostate cancers were above the normal tissue

standard. ADAM9, A disintegrin and metalloprotease 9.

3. Results

3.1. Quantitative reverse transcriptase-PCR

The normalised ADAM9 expression was signifi-cantly higher in malignant samples (n = 25) thanin matched nonmalignant samples (both wereevaluated for the total and transmembrane ADAM9),whereas the differences for the secreted form of

ADAM9 remained insignificant (Fig. 1A and B). Stillfor three (12%, total), five (20%, transmembrane),and eight (32%, secreted) samples, ADAM9 expres-sion levels were below those of the correspondingnormal tissue. ADAM9 mRNA expression did notcorrelate with any of the clinicopathological para-meters.

3.2. Western blot

Fig. 2A illustrates a representative blot of ADAM9protein expression in normal and tumour prostatetissue extracts. ADAM9 protein was detectable in alltissue extracts. With the use of Western blotanalysis under reducing conditions, two forms at

Fig. 2 – Western blots and antibody specificity testing. (A) ADAM9 and b-actin in normal (N) and tumour (T) prostate tissue

extracts. (B) Specificity of ADAM9 bands was confirmed with the use of a blocking peptide, corresponding to the carboxy

terminal end of human ADAM9. (C) Peptide blocking of the ADAM9 antibody for immunohistochemistry in corresponding

tissue sections. The figure demonstrates regular ADAM9 expression for the unblocked antibody (left part) and loss of

ADAM9 staining after peptide blocking (right part). ADAM9, A disintegrin and metalloprotease 9.

Table 3 – The amount of ADAM9 protein (Western blot) incancer tissue in relation to normal tissue

Patient Tumour (% density) Tumour (% density)

Total ADAM9(80 + 70 kDa)

80 kDaband

70 kDaband

1 134 183 79

2 84 0 96

3 178 100a 100

4 120 154 89

5 103 37 77

6 118 179 82

Median 119b 127 89

ADAM9, A disintegrin and metalloprotease 9.

Mean values of three independent Western blots. The ADAM9 level

of the normal tissue sample was set to 100%.a In the normal tissue of this patient, the 80 kDa band was absent.b Wilcoxon test (paired samples), p = 0.09.

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80 and 70 kDa were detected. In the presence ofexcess blocking peptide, all bands were stronglyreduced (Fig. 2B). In addition, none of these proteinswas visible after immunoblotting with control IgG ofserum from healthy nonimmunised rabbits, demon-strating antibody specificity of the two bands.Relative levels of the 80-kDa protein were increasedin prostate cancer compared with normal tissue (3 of6). The 80-KDa band was absent in two normal tissueextracts. In the cancer sample one of these patientsdisplayed a distinct band at this molecular weight.The expression of the 70-kDa ADAM9-like band wasdecreased in the prostate cancer extracts (3 of 6). Theexpression level of the total ADAM9 protein (80 and70 kDa together) is increased in prostate cancertissue compared with normal prostate tissue (4 of 6)(Table 3).

3.3. ADAM9 immunostaining in prostate tissues

ADAM9 was found in cancerous and normal pro-static epithelia. Of the prostate cancers adjacentnormal prostate tissue, 85.9% (170 of 198) and 88.4%(175 of 198), respectively, showed at least someADAM9 expression. Only 28 (14.1%) prostate cancerswere completely negative. An H-score below that ofthe normal tissue was detected for 50 cases (25.3%),

which is in line with the results from the RNAanalysis. Antibody specificity was confirmed bypeptide blocking (Fig. 2C). In cancerous glands adiffuse cytoplasmic immunoreactivity predomi-nated, whereas a minority of cases (63 cases,31.8%) displayed a luminally accentuated glycoca-lyceal staining pattern, as expected of a transmem-brane protein and as seen in epithelia of benignprostate glands (Fig. 3). Stromal tissue, peripheral

Table 4 – Correlation of ADAM9 protein expression inprostate cancer with conventional clinical or tumourparameters

ADAM9 pTstatus

Gleasonsum

Pre-OPPSA

Age R status

CC 0.039 0.074 �0.047 �0.079 0.007

p 0.585 0.301 0.542 0.270 0.921

N 198 197 169 198 196

ADAM9, A disintegrin and metalloprotease 9; pT status, tumour

stage; Pre-Op PSA, preoperative PSA; R status, residual tumour; CC,

correlation coefficient; p, two-sided significance; N, number of

cases.

Fig. 3 – Immunohistological expression of ADAM9. (A–C) Glandular prostate cancer tissue displaying weak (A), moderate (B),

and strong (C) ADAM9 protein expression. (D) Prostatic intraepithelial neoplasia (small arrow) surrounded by microglandular

prostate cancer (bold arrows) and an adjacent normal prostatic gland (unfilled arrow). (E and F) Most cases displayed a rather

diffuse cytoplasmic staining for ADAM9 (E, cancer = bold arrow, normal gland = unfilled arrow), but a minority of cases

showed a marked accentuation of a luminally polarised ADAM9 expression (F). ADAM9, A disintegrin and metalloprotease 9.

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nerves, and peripheral vessels were negative forADAM9, whereas skeletal muscle cells as well asganglion cells were positive.

The median H-scores of prostate cancer, PIN(n = 60), and normal tissue were 100, 100, and 90,respectively. The difference in the ADAM9 proteinexpression of tumour and normal tissue was highlysignificant ( p < 0.001). PIN was present in 60 (30.3%)of the 198 cases. ADAM9 expression of PIN wassignificantly higher ( p = 0.001) than in normaltissue but did not differ significantly from theADAM9 expression in the invasive carcinoma( p = 0.645).

Table 5 – Univariate survival analysis (Kaplan-Meier): PSA relapse times of all patients with prostate according toclinicopathological factors and ADAM9 expression

Characteristic No. of cases No. of events 3-year PSA relapse rate (�SE) in % p value

ADAM9 expression 0.003

low 85 19 14.9 � 4.0

high 113 50 28.9 � 4.4

Pre-OP PSA 0.007

�9.4 ng/ml 87 22 22.8 � 4.6

>9.4 ng/ml 82 38 27.3 � 5.1

Age 0.168

�62 yr 105 40 26.1 � 4.4

>62 yr 93 29 19.3 � 4.2

pT status <0.001

pT2 110 27 15.4 � 3.6

pT3/4 88 42 31.8 � 5.1

Gleason sum <0.001

3–6 89 19 11.7 � 3.5

7 56 18 24.7 � 6.0

7–10 52 32 41.1 � 7.1

Residual tumour 0.001

R0 109 29 17.4 � 3.7

R1 87 39 30.0 � 5.0

PSA, prostate-specific antigen; ADAM9, A disintegrin and metalloprotease 9; SE, standard error; Pre-Op PSA, preoperative PSA; pT status,

tumour stage.

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3.4. ADAM9 expression, clinicopathological correlations,

and disease-free survival times

In bivariate correlations, the ADAM9 protein expres-sion in prostate cancer did not correlate with any ofthe clinicopathological parameters (Table 4). In thechi-square tests, higher ADAM9 protein expressionwas significantly associated with younger age(Table 1). In univariate survival analysis (Kaplan-Meier) of PSA relapse-free survival times, preopera-tive PSA, pT status (tumour grade), Gleason score,and residual tumour status reached statisticalsignificance (Table 5). Higher ADAM9 expressionin prostate cancer was significantly associated withshortened PSA relapse-free survival times (Fig. 4A,p = 0.003).

To evaluate the influence of the intracellularADAM9 distribution (diffuse cytoplasmic vs. lumin-ally accentuated - polar), we stratified the formeranalysis. The resulting Kaplan-Meier curve (Fig. 4B)revealed a significantly longer PSA relapse-freesurvival for those patients with low ADAM9 andpolar staining pattern. High ADAM9 expressionwas generally unfavourable, but even low ADAM9expression was disadvantageous if diffuse.

After stratification according to antiandrogenicpretreatment, total ADAM9 lost much of its prog-nostic value in patients not pretreated and remaineda significant prognosticator in the group of pre-treated patients only (Fig. 4C). However, when the

ADAM9 distribution patterns were considered,patients with tumours displaying a low polarADAM9 expression had a significantly more favour-able course irrespective of antiandrogen pretreat-ment. We suspected a systemic bias in the group ofpatients who had received antiandrogenic pretreat-ment because this group of patients had (in themedian) higher Gleason scores and higher pT stages.Therefore, a subcohort of patients without pretreat-ment but matching Gleason scores and pT stageswas compiled and subsequently analysed. In thisgroup, no prognostic value of total ADAM9 wasapparent (53 vs. 61 mo, p = 0.455), which suggests afunctional link between androgen ablation andADAM9 expression.

A Cox multivariate analysis over all cases includ-ing pT status, Gleason sum, preoperative PSA level,and residual tumour status further confirmed theindependent prognostic value of total ADAM9(Table 6). This finding was also true for antiandro-gen-pretreated patients in whom ADAM9 was theonly significant parameter for PSA relapse (relativerisk, 3.607; p = 0.031).

4. Discussion

ADAM9 overexpression has been described in awealth of solid tumours including prostate cancer[15,18–21]. In gastric cancer ADAM9 is significantly

Fig. 4 – Prostate-specific antigen (PSA) relapse-free survival curve for ADAM9. The number of events (PSA relapses) in the

specific subgroup is given in brackets. (A) Significantly shorter PSA relapse-free survival times for patients with higher

ADAM9 protein expression. (B) Kaplan-Meier curves for high versus low ADAM9 expression stratified for intracellular

distribution patterns. (C) Kaplan-Meier curve for the subgroup of antiandrogen pretreated patients. ADAM9, A disintegrin

and metalloprotease 9.

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upregulated and ADAM9 inhibition resulted ininhibited cell growth of gastric cancer cell lines[22]. ADAM9 transcript upregulation was found inconditions associated with liver injury [23]. Grutz-mann et al [24] and Alldinger et al [25] discoveredADAM9 mRNA upregulated in pancreatic ductaladenocarcinoma and confirmed an ADAM9 over-

Table 6 – Multivariate survival analysis (Cox regressionmodel) for ADAM9, pT status, Gleason sum, preoperativePSA, and residual tumour status

Variable Relative risk 95%CI p value

ADAM9 1.871 1.030–3.397 0.040

PT status 1.305 0.687–2.479 0.416

Gleason sum 1.719 1.183–2.497 0.004

Pre-OP PSA 1.642 0.959–2.813 0.071

Residual tumour 1.830 0.833–2.702 0.176

ADAM9, A disintegrin and metalloprotease 9; CI, confidence

interval; pT status, tumour stage; Pre-OP PSA, preoperative PSA.

expression in 70% of pancreatic carcinomas byimmunohistochemistry. In a following study of thisgroup, a significant prognostic value of ADAM9expression was demonstrated, which matches ourfindings in prostate cancer [17]. O’Shea et al [19]investigated ADAM9 mRNA and protein expressionin breast cancer, and detected ADAM9 mRNA andthe mature protein significantly upregulated andassociated with a positive nodal status and over-expression of HER-2/neu protein [19]. As there weredifferences in the levels of mature ADAM9 and theprecursor form of the protein between cancer andbenign lesions, an alternative processing of ADAM9in normal and cancerous tissue was suggested.Interestingly, the same study demonstrated ADAM9mRNA in two of two prostate cancer cases [19]. Chinet al [26] analysed genome copy number abnorm-alities in 145 breast cancers and found ADAM9among nine highly amplified genes, which were, onthe basis of supposed protein characteristics [27],

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considered druggable. Overexpression of ADAM9 inlung cancer cell lines resulted in enhanced inva-siveness and was significantly related to brainmetastasis [13]. In melanoma ADAM9 is upregulatedin vivo at the invasion front, further supporting itsrole for tumour progression [28]. Next to thetransmembrane form of ADAM9, a secreted formthat might be relevant for tumour progression hasbeen described [8,29]. We analysed the transmem-brane and the supposedly secreted form of ADAM9by quantitative reverse transcriptase-PCR and couldconfirm the upregulation of total ADAM9. Usingprimers specific for the transmembrane and thesecreted form, we found that both were upregulated,although statistical significance was for only thetransmembrane form, which might be due to oursmall sample size (n = 25).

To our knowledge Karan et al [18] were the first todescribe ADAM9 expression in prostate cancer. Theyfound ADAM9 expression in nine prostate cancercell lines and in eight out of eight histologicalprostate cancer samples, a finding we confirmedpreviously [15]. We found ADAM9 protein signifi-cantly upregulated in cancerous tissue comparedwith adjacent normal tissue, which was alsomirrored by the mRNA results. Still, in up to around30% of tumours, ADAM9 was downregulated.Immunohistochemically we observed different pat-terns of ADAM9 immunoreactivity. In normalprostate glands, we found a luminally polarisedorientation of ADAM9, which was also seen in aminority of tumours, albeit with a higher intensity.However, in most tumours a depolarised diffusecytoplasmic immunoreactivity was noted, whichwas not correlated with Gleason score. The stainingpatterns were not associated with different prog-nostic values, but showed considerable prognosticimpact, if combined with low or high levels ofADAM9 expression. Importantly, patients showing alow polarised ADAM9 expression in their tumours(comparable to the ADAM9 pattern in normalglands) have a comparatively excellent prognosis.Peduto et al [20] found that ADAM9 could cleave andrelease EGF, and observed a positive association ofADAM9 with larger and less well-differentiatedprostate tumours. Although this is a finding thatcould not be verified in our study cohort, our dataclearly show a negative prognostic impact of higherADAM9 levels, indicating a more aggressive disease.This finding is in line with Sung et al [21] whoshowed elevated ADAM9 levels during the transitionfrom androgen-dependent to androgen-indepen-dent and metastatic cancer cells, using a tissuemicroarray (n = 200), which apparently did not allowa detailed survival analysis.

Sung et al [21] also found an inducibility ofADAM9 by oxidative stress and reactive oxygenspecies in prostate cancer cell lines and suggested,in line with Fisher et al [9], that ADAM9 would conferresistance to stress induced injuries. The induci-bility of ADAM9 appeared to be pronounced inandrogen-dependent tumours [30]. This functionaldata might be of relevance to our observations of theprognostic value of ADAM9 in antiandrogenicallypretreated patients. It can be hypothesised thattumour cells that can mediate the stress induced byandrogen ablation by ADAM9 upregulation have asurvival benefit and are thus more aggressive. Itremains to be shown in larger cohorts if ADAM9expression can indeed be used as a predictivemarker of response to antiandrogenic therapy inprostate cancer.

In conclusion our results support the generalnotion of ADAM9 to be associated with moreaggressive tumour behaviour in prostate cancer.ADAM9 protein expression was significantly andindependently associated with shortened PSArelapse-free survival, which suggests its use as aprognostic marker in prostate cancer followingfurther validation.

Conflicts of interest

The authors have nothing to disclose.

Acknowledgements

We are grateful to Britta Beyer for excellent technicalassistance. The support of Ilka Kristiansen and DrMarius Fritzsche is greatly acknowledged.

Appendix A. Supplementary data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.eururo.2007.11.034 and via www.europeanurology.com.

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factor. EMBO J 1998;17:7260–72.

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ADAM9 enhances growth factor-mediated recycling of

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gastric cancer cells. Gastroenterology 2004;127:559–69.

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of ADAM9 in non-small cell lung cancer correlates with

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Editorial Comment on: ADAM9 Expression is aSignificant and Independent Prognostic Markerof PSA Relapse in Prostate CancerAlessandra Valentini, Sergio BernardiniDepartment of Internal Medicine, University of Rome‘‘Tor Vergata’’, Rome, Italybernardini@med.uniroma2.it

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inhibitor of metalloproteinase-3) in human prostatic ade-

nocarcinomas. Int J Oncol 2003;23:1365–71.

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mRNA and protein in human breast cancer. Int J Cancer

2003;105:754–61.

[20] Peduto L, Reuter VE, Shaffer DR, Scher HI, Blobel CP.

Critical function for ADAM9 in mouse prostate cancer.

Cancer Res 2005;65:9312–9.

[21] Sung SY, Kubo H, Shigemura K, et al. Oxidative stress

induces ADAM9 protein expression in human prostate

cancer cells. Cancer Res 2006;66:9519–26.

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C. The disintegrin-metalloproteinases ADAM9, ADAM12,

and ADAM15 are upregulated in gastric cancer. Int J Oncol

2005;26:17–24.

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human liver cancers: TGF-beta-regulated expression in

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sion profiles of microdissected pancreatic ductal adeno-

carcinoma. Virchows Arch 2003;443:508–17.

[25] Alldinger I, Dittert D, Peiper M, et al. Gene expression

analysis of pancreatic cell lines reveals genes overex-

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[26] Chin K, DeVries S, Fridlyand J, et al. Genomic and tran-

scriptional aberrations linked to breast cancer pathophy-

siologies. Cancer Cell 2006;10:529–41.

[27] Russ AP, Lampel S. The druggable genome: an update.

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[28] Zigrino P, Mauch C, Fox JW, Nischt R. Adam-9 expression

and regulation in human skin melanoma and melanoma

cell lines. Int J Cancer 2005;116:853–9.

[29] Mazzocca A, Coppari R, De Franco R, et al. A secreted form

of ADAM9 promotes carcinoma invasion through tumor-

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[30] Shigemura K, Sung SY, Kubo H, et al. Reactive oxygen

species mediate androgen receptor- and serum starva-

tion-elicited downstream signaling of ADAM9 expression

in human prostate cancer cells. Prostate 2007;67:722–31.

Epidermal growth factor, insulin-like growthfactor, and mutations of androgen receptor play arole in the progression of hormone-sensitiveprostate cancer to hormone insensitivity.

Although hormonal therapy is used earlier in thecourse of disease and a primary response rate of80–90% occurs with hormonal ablation, almost all

patients advance to a state of androgen indepen-dence manifested by increasing prostate-specificantigen (PSA) levels and new lesions on bone scans.The median survival time in patients with andro-gen-independent prostate cancer is about 18 moand this condition is also accompanied by theattainment of high resistance to cytotoxic drugs [1].Androgen independence may be an intrinsic, butresting, property of some prostate cancer cells thatare activated in response to androgen deprivation.Only epithelial cells, not basal or stromal cells,undergo apoptosis on androgen withdrawal. Theclonal selection hypothesis relates to the selectivesurvival of preexisting androgen-independent cellswithin the tumour by hormonal deprivation [2].These clonal cells are thought to be poorlydifferentiated and similar to basal or stem cells [3].

Substantial attempts have been expended indescribing the available factors and determiningtheir predictive value for staging, cancer recur-rence, survival, and androgen-independent devel-opment. Interesting observations are constantlybeing made concerning the expression of a widerange of cellular components, including enzymes,structural proteins, ion channels, ligand receptors,gene sequences, among others, that are associatedwith progression of prostate cancer.

Gene products of the A disintegrin and metallo-proteinase (ADAM) family are critically involved incarcinogenesis and tumour progression of varioussolid tumours. It has been described that prostatetumours revealed high levels of ADAM9 mRNA inwell-differentiated carcinomas, but only low orundetectable levels in poorly differentiated carci-nomas. The loss-of-function experiments suggestthat ADAM9 is critical for tumour progression pastthe well-differentiated state, whereas gain-of-function experiments provided additional evi-dence for a causal role of ADAM9 in tumorigenesis.

ADAM9 deficiency delays or prevents prostatetumour progression to advanced states in a mouseprostate cancer model, whereas deregulatedexpression of ADAM9 initially disrupts epithelial

stromal homeostasis in the prostate epithelium,and later leads to prostatic intraepithelial neopla-sia lesions [4]. Interestingly, Fritzsche et al foundthat ADAM9 was significantly up-regulated inprostate cancer in comparison to normal tissueon mRNA and protein levels. ADAM9 proteinexpression was significantly associated with shor-tened PSA relapse-free survival in univariate andmultivariate analyses, particularly in patients whohad received prior androgen ablation [5].

Additionally, the authors hypothesise thatADAM9 is a predictive marker of response toantiandrogenic therapy. Thus, this last hypothesisseems to be more interesting than the single valueof ADAM9 as an independent prognostic factor,to individualise the gene as a molecular markerfor hormone-independent switching of prostatecancer.

References

[1] Yagoda A, Petrylak D. Cytotoxic chemotherapy for

advanced hormone-resistant prostate cancer. Cancer

1993;71(Suppl 3):1098–109.

[2] Isaacs JT, Coffey DS. Adaptation versus selection as the

mechanism responsible for the relapse of prostatic can-

cer to androgen ablation therapy as studied in the Dun-

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[3] Rashid MH, Chaudhary UB. Intermittent androgen depri-

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295–301.

[4] Peduto L, Reuter VE, Shaffer DR, Scher HI, Blobel CP.

Critical function for ADAM9 in mouse prostate cancer.

Cancer Res 2005;20:9312–8.

[5] Fritzsche FR, Jung M, Tolle A, et al. ADAM9 expression is

a significant and independent prognostic marker of PSA

relapse in prostate cancer. Eur Urol 2008;54:1097–108.

DOI: 10.1016/j.eururo.2007.11.035

DOI of original article: 10.1016/j.eururo.2007.11.034

Editorial Comment on: ADAM9 Expression is aSignificant and Independent Prognostic Markerof PSA Relapse in Prostate CancerColin CooperInstitute of Cancer Research, Sutton, UKColin.Cooper@icr.ac.uk

ADAM9 (a disintegrin and metalloprotease-9) is amembrane-anchored metalloproteinase that playsa key role in the ectodermal shedding of growthfactors. Many growth factors and cytokines, includ-ing all ligands for epidermal growth factor receptor,are synthesized as membrane-anchored proteins.

e u r o p e a n u r o l o g y 5 4 ( 2 0 0 8 ) 1 0 9 7 – 1 1 0 8 1107

ADAM9 and other members of this family areresponsible for catalyzing their release from thecell by ectodermal shedding, a process vital for cell-to-cell communication and for cellular homeosta-sis. ADAM9 is also a cell adhesion molecule thatinteracts with the integrin avb5 and that may beinvolved in controlling cell invasion. The elevatedlevels of this protein observed in many commonneoplasms, including prostate, suggests a role forthis protein in the development or progression ofcancer, a view supported by the recent observationthat the ADAM9 gene is both amplified and over-expressed in human breast cancer [1]. In the W10

mouse prostate cancer model knocking out theADAM9 gene made the tumors that develop smallerand more differentiated in appearance, whereas ingain-of-function experiments overexpression ofADAM9 caused the development of prostatic intra-epithelial hyperplasia [2]. In this issue Fritzsche et al[3] provide further compelling evidence for theimportance of ADAM9 overexpression in determin-ing aggression in human prostate cancer. In a studyof 198 prostatectomy cases they showed thatADAM9 expression was significantly associatedwith shortened disease-free relapse. Importantly,in multivariate analyses the expression of ADAM9provided prognostic information in addition to thatobtained from stage, Gleason score, and preopera-tive prostate-specific antigen (PSA) level. Thisassociation still held when the authors consideredonly those patients treated with antiandrogentherapy prior to surgery. It has been shown in otherstudies that ADAM9 levels in prostate cancer cells

are controlled by androgens and by oxidative stressin such a way that induction of ADAM9 expressioncan be completely abrogated by the presence ofantioxidants [4,5]. These results raise the prospectthat the modulation of ADAM9 level or the devel-opment of drugs that directly target its catalyticactivity may be of use in managing prostatecancer.

References

[1] Peduto L, Reuter VE, Shaffer DR, Scher HI, Blobel CP.

Critical function for ADAM9 in mouse prostate cancer.

Cancer Res 2005;65:9312–9.

[2] Sung SY, Kubo H, Shigemura K, et al. Oxidative stress

induces ADAM9 protein expression in human prostate

cancer cells. Cancer Res 2006;66:9519–26.

[3] Fritzsche FR, Jung M, Tolle A, et al. ADAM9 expression is

a significant and independent prognostic marker of PSA

relapse in prostate cancer. Eur Urol 2008;54:1097–108.

[4] Shigemura K, Sung SY, Kubo H, et al. Reactive oxygen

species mediate androgen receptor- and serum starva-

tion-elicited downstream signaling of ADAM9 expres-

sion in human prostate cancer cells. Prostate 2007;67:

722–31.

[5] Chin K, DeVries S, Fridlyand J, et al. Genomic and tran-

scriptional aberrations linked to breast cancer patho-

physiologies. Cancer Cell 2006;10:529–41.

DOI: 10.1016/j.eururo.2007.11.036

DOI of original article: 10.1016/j.eururo.2007.11.034

e u r o p e a n u r o l o g y 5 4 ( 2 0 0 8 ) 1 0 9 7 – 1 1 0 81108