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

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

european urology 54 (2008) 1097–1108 available at www.sciencedirect.com journal homepage: www.europeanurology.com Prostate Cancer ADAM9 Expression ...

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european urology 54 (2008) 1097–1108

available at www.sciencedirect.com journal homepage: www.europeanurology.com

Prostate Cancer

ADAM9 Expression is a Significant and Independent Prognostic Marker of PSA Relapse in Prostate Cancer Florian R. Fritzsche a, Monika Jung b, Angelika To¨lle 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 Gru¨tzmann e, Klaus Jung b, Glen Kristiansen a,c,* a

Institute of Pathology, Charite´ - Universita¨tsmedizin Berlin, Berlin, Germany Department of Urology, Charite´ - Universita¨tsmedizin Berlin, Berlin, Germany c Institute of Pathology, Universita¨tsspital Zu¨rich, Zu¨rich, Switzerland d Institute of Pathology, University of Regensburg, Regensburg, Germany e Department of Surgery, University Hospital Dresden, Dresden, Germany b

Article info

Abstract

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

Objectives: A disintegrin and metalloprotease (ADAM) 9 has been implicated in tumour progression of prostate cancer. We evaluated the expression of ADAM9 on protein and messenger RNA (mRNA) level in a larger cohort of prostate cancer cases following prostatectomy and correlated the findings with clinicopathological parameters including prostate-specific antigen (PSA) relapse times. Methods: We immunostained 198 clinicopathologically characterised prostate cancer cases for ADAM9. For 25 additional cases, ADAM9 mRNA of microdissected tumour and normal tissue was analysed via quantitative reverse transcriptase-polymerase chain reaction. Results: ADAM9 was significantly upregulated in prostate cancer compared with normal tissue on mRNA and protein level. ADAM9 protein expression was significantly associated with shortened PSA relapse-free survival in univariate and multivariate analyses, particularly in patients who had received prior androgen ablation. Conclusions: ADAM9 is overexpressed in prostate cancer cases and is an independent prognostic marker of PSA relapse-free survival following radical prostatectomy. Further studies are needed to verify its role as a predictive marker of response to androgen ablation.

Keywords: ADAM9 Immunohistochemistry qRT-PCR Prognostic marker Prostate cancer

# 2007 European Association of Urology. Published by Elsevier B.V. All rights reserved. * 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: [email protected] (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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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 is involved in different physiological and pathophysiological functions including cell adhesion, cell migration, and tissue remodeling [1]. ADAMs are membrane-anchored glycoproteins that consist of a protease and an adhesion domain. The interactions of ADAMs with cell-surface and extracellular matrix proteins could be of relevance to tumour biology because these processes are vital for tumour progression [2–4]. Concordantly, various ADAMs have been shown to represent a potential diagnostic and prognostic marker in human tumours [5]. The type I transmembrane protein ADAM9 (synonyms, MDC9, meltrin-g) is a cell adhesion molecule, found to interact with avb5 integrin, to cleave the insulin B-chain, and to be involved in the ectodomain shedding of membrane-anchored heparin-binding epidermal growth factor (EGF)-like growth factor [4,6,7]. In addition, a secreted form of ADAM9 that resulted from alternative splicing was described [8]. ADAM9 might be essentially involved in carcinogenesis and tumour progression by mediating EGF receptor activity and by promoting cancer

cell invasion via regulation of E-cadherin and integrins [6,9–15]. In this study we evaluated ADAM9 expression on protein and transcript level to clarify a diagnostic or prognostic value of ADAM9 in human prostate cancer. 2.

Methods

2.1. Patients (reverse transcriptase-polymerase chain reaction) Twenty-five matched (tumour/normal) prostate samples were microdissected from frozen sections. Cases used for microdissection 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 hormone 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 morphologically discernable effects, Gleason score was evaluated as

Table 1 – Associations (chi-square tests) between the protein expression of ADAM9 in prostate cancer and clinicopathological parameters

All cases

Total

Low ADAM9a

High ADAM9a

198

85 (42.9%)

113 (57.1%)

105 93

36 (34.3%) 49 (52.7%)

69 (65.7%) 44 (47.3%)

87 82

35 (40.2%) 34 (41.5%)

52 (59.8%) 48 (58.5%)

110 88

50 (45.5%) 35 (39.8%)

60 (54.5%) 53 (60.2%)

89 56 52

40 (44.9%) 24 (42.9%) 21 (40.4%)

49 (55.1%) 32 (57.1%) 31 (59.6%)

109 87

48 (44.0%) 35 (40.2%)

61 (56.0%) 52 (59.8%)

Age

0.010 62 >62

Pre-OP PSAb

0.877 9.4 ng/ml >9.4 ng/ml

PT status

0.471 pT2 pT3/4c

Gleason sumd

0.597 3–6 7 8–10

Residual tumoure

0.663 R0 R1

ADAM9, A disintegrin and metalloprotease 9; Pre-Op PSA, preoperative PSA; pT status, tumour stage. 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. a

p value

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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 microdissected areas of normal prostate tissue, prostatic intraepithelial 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, Wilmington, 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 PCRspecific amplicons. For all genes, calibration curves with

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 calculated 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 prostatectomy specimens were used for protein extraction. After histological control to ascertain the specific histology the tissue was frozen, minced, and homogenised by an ultrasound 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 aprotonin, 10 mg/ml soybean trypsin inhibitor). After centrifugation 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

Table 2 – Real-time reverse transcriptase-PCR data with primer sequences Target gene and transcript variants

ACC no.

Primer sequence 50 . . .. . .30

PS (bp)

E

Total ADAM 9

NM_003816

F: cag atg gca aaa atc aag ca R: gat ggg aac tgc tga ggt tg

151

2.01

Transcript variant 1 (transmembrane)

NM_003816

F: gga tac gga gga agt gtg ga R: gca cag aca ata agg gga aca

110

1.91

Transcript variant 2 (cytosolic)

NM_001005845

F: gtc atg gac atg gga aat ga R: ctc cac agt tga tcc ctc ttg

120

1.95

PCR, polymerase chain reaction; ACC no, GenBank accession number; PS, PCR product sizes; E, efficiencies of target gene PCRs; ADAM9, A disintegrin and metalloprotease 9.

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antibody, and (b) control IgG of serum from healthy nonimmunised rabbits (Dako).

2.5.

Immunohistochemistry

Formalin-fixed paraffin-embedded tissue was freshly cut (3 mm), mounted on superfrost slides (Menzel Gla¨ser), 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 immunoreactivity. As negative controls we used ADAM9-positive prostate cancer cases omitting the primary antibody.

2.6.

ADAM9 remained insignificant (Fig. 1A and B). Still for three (12%, total), five (20%, transmembrane), and eight (32%, secreted) samples, ADAM9 expression levels were below those of the corresponding normal tissue. ADAM9 mRNA expression did not correlate with any of the clinicopathological parameters. 3.2.

Western blot

Fig. 2A illustrates a representative blot of ADAM9 protein expression in normal and tumour prostate tissue extracts. ADAM9 protein was detectable in all tissue extracts. With the use of Western blot analysis under reducing conditions, two forms at

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 fourtier 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.

3.

Results

3.1.

Quantitative reverse transcriptase-PCR

The normalised ADAM9 expression was significantly higher in malignant samples (n = 25) than in matched nonmalignant samples (both were evaluated for the total and transmembrane ADAM9), whereas the differences for the secreted form of

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.

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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.

80 and 70 kDa were detected. In the presence of excess blocking peptide, all bands were strongly reduced (Fig. 2B). In addition, none of these proteins was visible after immunoblotting with control IgG of serum from healthy nonimmunised rabbits, demonstrating antibody specificity of the two bands. Relative levels of the 80-kDa protein were increased in prostate cancer compared with normal tissue (3 of 6). The 80-KDa band was absent in two normal tissue extracts. In the cancer sample one of these patients displayed a distinct band at this molecular weight. The expression of the 70-kDa ADAM9-like band was decreased in the prostate cancer extracts (3 of 6). The expression level of the total ADAM9 protein (80 and 70 kDa together) is increased in prostate cancer tissue compared with normal prostate tissue (4 of 6) (Table 3). 3.3.

ADAM9 immunostaining in prostate tissues

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

which is in line with the results from the RNA analysis. Antibody specificity was confirmed by peptide blocking (Fig. 2C). In cancerous glands a diffuse cytoplasmic immunoreactivity predominated, whereas a minority of cases (63 cases, 31.8%) displayed a luminally accentuated glycocalyceal staining pattern, as expected of a transmembrane protein and as seen in epithelia of benign prostate glands (Fig. 3). Stromal tissue, peripheral Table 3 – The amount of ADAM9 protein (Western blot) in cancer tissue in relation to normal tissue Patient

1 2 3 4 5 6 Median

Tumour (% density)

Tumour (% density)

Total ADAM9 (80 + 70 kDa)

80 kDa band

134 84 178 120 103 118 119b

183 0 100a 154 37 179 127

70 kDa band 79 96 100 89 77 82 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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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.

nerves, and peripheral vessels were negative for ADAM9, whereas skeletal muscle cells as well as ganglion 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 protein expression of tumour and normal tissue was highly significant ( p < 0.001). PIN was present in 60 (30.3%) of the 198 cases. ADAM9 expression of PIN was significantly higher ( p = 0.001) than in normal tissue but did not differ significantly from the ADAM9 expression in the invasive carcinoma ( p = 0.645).

Table 4 – Correlation of ADAM9 protein expression in prostate cancer with conventional clinical or tumour parameters ADAM9

pT status

Gleason sum

Pre-OP PSA

Age

CC p N

0.039 0.585 198

0.074 0.301 197

0.047 0.542 169

0.079 0.270 198

R status 0.007 0.921 196

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.

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

No. of cases

No. of events

3-year PSA relapse rate (SE) in %

ADAM9 expression low high

85 113

19 50

14.9  4.0 28.9  4.4

Pre-OP PSA 9.4 ng/ml >9.4 ng/ml

87 82

22 38

22.8  4.6 27.3  5.1

Age 62 yr >62 yr

105 93

40 29

26.1  4.4 19.3  4.2

pT status pT2 pT3/4

110 88

27 42

15.4  3.6 31.8  5.1

Gleason sum 3–6 7 7–10

89 56 52

19 18 32

11.7  3.5 24.7  6.0 41.1  7.1

Residual tumour R0 R1

109 87

29 39

17.4  3.7 30.0  5.0

p value 0.003

0.007

0.168

<0.001

<0.001

0.001

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

3.4. ADAM9 expression, clinicopathological correlations, and disease-free survival times

In bivariate correlations, the ADAM9 protein expression in prostate cancer did not correlate with any of the clinicopathological parameters (Table 4). In the chi-square tests, higher ADAM9 protein expression was significantly associated with younger age (Table 1). In univariate survival analysis (KaplanMeier) of PSA relapse-free survival times, preoperative PSA, pT status (tumour grade), Gleason score, and residual tumour status reached statistical significance (Table 5). Higher ADAM9 expression in prostate cancer was significantly associated with shortened PSA relapse-free survival times (Fig. 4A, p = 0.003). To evaluate the influence of the intracellular ADAM9 distribution (diffuse cytoplasmic vs. luminally accentuated - polar), we stratified the former analysis. The resulting Kaplan-Meier curve (Fig. 4B) revealed a significantly longer PSA relapse-free survival for those patients with low ADAM9 and polar staining pattern. High ADAM9 expression was generally unfavourable, but even low ADAM9 expression was disadvantageous if diffuse. After stratification according to antiandrogenic pretreatment, total ADAM9 lost much of its prognostic value in patients not pretreated and remained a significant prognosticator in the group of pretreated patients only (Fig. 4C). However, when the

ADAM9 distribution patterns were considered, patients with tumours displaying a low polar ADAM9 expression had a significantly more favourable course irrespective of antiandrogen pretreatment. We suspected a systemic bias in the group of patients who had received antiandrogenic pretreatment because this group of patients had (in the median) higher Gleason scores and higher pT stages. Therefore, a subcohort of patients without pretreatment but matching Gleason scores and pT stages was compiled and subsequently analysed. In this group, no prognostic value of total ADAM9 was apparent (53 vs. 61 mo, p = 0.455), which suggests a functional link between androgen ablation and ADAM9 expression. A Cox multivariate analysis over all cases including pT status, Gleason sum, preoperative PSA level, and residual tumour status further confirmed the independent prognostic value of total ADAM9 (Table 6). This finding was also true for antiandrogen-pretreated patients in whom ADAM9 was the only significant parameter for PSA relapse (relative risk, 3.607; p = 0.031).

4.

Discussion

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

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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.

upregulated and ADAM9 inhibition resulted in inhibited cell growth of gastric cancer cell lines [22]. ADAM9 transcript upregulation was found in conditions associated with liver injury [23]. Gru¨tzmann et al [24] and Alldinger et al [25] discovered ADAM9 mRNA upregulated in pancreatic ductal adenocarcinoma and confirmed an ADAM9 over-

Table 6 – Multivariate survival analysis (Cox regression model) for ADAM9, pT status, Gleason sum, preoperative PSA, and residual tumour status Variable ADAM9 PT status Gleason sum Pre-OP PSA Residual tumour

Relative risk

95%CI

1.871 1.305 1.719 1.642 1.830

1.030–3.397 0.687–2.479 1.183–2.497 0.959–2.813 0.833–2.702

p value 0.040 0.416 0.004 0.071 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 by immunohistochemistry. In a following study of this group, a significant prognostic value of ADAM9 expression was demonstrated, which matches our findings in prostate cancer [17]. O’Shea et al [19] investigated ADAM9 mRNA and protein expression in breast cancer, and detected ADAM9 mRNA and the mature protein significantly upregulated and associated with a positive nodal status and overexpression of HER-2/neu protein [19]. As there were differences in the levels of mature ADAM9 and the precursor form of the protein between cancer and benign lesions, an alternative processing of ADAM9 in normal and cancerous tissue was suggested. Interestingly, the same study demonstrated ADAM9 mRNA in two of two prostate cancer cases [19]. Chin et al [26] analysed genome copy number abnormalities in 145 breast cancers and found ADAM9 among nine highly amplified genes, which were, on the basis of supposed protein characteristics [27],

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considered druggable. Overexpression of ADAM9 in lung cancer cell lines resulted in enhanced invasiveness and was significantly related to brain metastasis [13]. In melanoma ADAM9 is upregulated in vivo at the invasion front, further supporting its role for tumour progression [28]. Next to the transmembrane form of ADAM9, a secreted form that might be relevant for tumour progression has been described [8,29]. We analysed the transmembrane and the supposedly secreted form of ADAM9 by quantitative reverse transcriptase-PCR and could confirm the upregulation of total ADAM9. Using primers specific for the transmembrane and the secreted form, we found that both were upregulated, although statistical significance was for only the transmembrane form, which might be due to our small sample size (n = 25). To our knowledge Karan et al [18] were the first to describe ADAM9 expression in prostate cancer. They found ADAM9 expression in nine prostate cancer cell lines and in eight out of eight histological prostate cancer samples, a finding we confirmed previously [15]. We found ADAM9 protein significantly upregulated in cancerous tissue compared with adjacent normal tissue, which was also mirrored by the mRNA results. Still, in up to around 30% of tumours, ADAM9 was downregulated. Immunohistochemically we observed different patterns of ADAM9 immunoreactivity. In normal prostate glands, we found a luminally polarised orientation of ADAM9, which was also seen in a minority of tumours, albeit with a higher intensity. However, in most tumours a depolarised diffuse cytoplasmic immunoreactivity was noted, which was not correlated with Gleason score. The staining patterns were not associated with different prognostic values, but showed considerable prognostic impact, if combined with low or high levels of ADAM9 expression. Importantly, patients showing a low polarised ADAM9 expression in their tumours (comparable to the ADAM9 pattern in normal glands) have a comparatively excellent prognosis. Peduto et al [20] found that ADAM9 could cleave and release EGF, and observed a positive association of ADAM9 with larger and less well-differentiated prostate tumours. Although this is a finding that could not be verified in our study cohort, our data clearly show a negative prognostic impact of higher ADAM9 levels, indicating a more aggressive disease. This finding is in line with Sung et al [21] who showed elevated ADAM9 levels during the transition from androgen-dependent to androgen-independent and metastatic cancer cells, using a tissue microarray (n = 200), which apparently did not allow a detailed survival analysis.

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Sung et al [21] also found an inducibility of ADAM9 by oxidative stress and reactive oxygen species in prostate cancer cell lines and suggested, in line with Fisher et al [9], that ADAM9 would confer resistance to stress induced injuries. The inducibility of ADAM9 appeared to be pronounced in androgen-dependent tumours [30]. This functional data might be of relevance to our observations of the prognostic value of ADAM9 in antiandrogenically pretreated patients. It can be hypothesised that tumour cells that can mediate the stress induced by androgen ablation by ADAM9 upregulation have a survival benefit and are thus more aggressive. It remains to be shown in larger cohorts if ADAM9 expression can indeed be used as a predictive marker of response to antiandrogenic therapy in prostate cancer. In conclusion our results support the general notion of ADAM9 to be associated with more aggressive tumour behaviour in prostate cancer. ADAM9 protein expression was significantly and independently associated with shortened PSA relapse-free survival, which suggests its use as a prognostic marker in prostate cancer following further validation.

Conflicts of interest The authors have nothing to disclose.

Acknowledgements We are grateful to Britta Beyer for excellent technical assistance. The support of Ilka Kristiansen and Dr Marius Fritzsche is greatly acknowledged.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j. eururo.2007.11.034 and via www.europeanurology. com. References [1] Nath D, Slocombe PM, Webster A, et al. Meltrin gamma (ADAM-9) mediates cellular adhesion through alpha(6)beta(1)integrin, leading to a marked induction of fibroblast cell motility. J Cell Sci 2000;113(pt 12):2319–28. [2] Iba K, Albrechtsen R, Gilpin BJ, Loechel F, Wewer UM. Cysteine-rich domain of human ADAM 12 (meltrin alpha)

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[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

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supports tumor cell adhesion. Am J Pathol 1999;154:1489– 501. Schwettmann L, Tschesche H. Cloning and expression in Pichia pastoris of metalloprotease domain of ADAM 9 catalytically active against fibronectin. Protein Expr Purif 2001;21:65–70. Zhou M, Graham R, Russell G, Croucher PI. MDC-9 (ADAM9/Meltrin gamma) functions as an adhesion molecule by binding the alpha(v)beta(5) integrin. Biochem Biophys Res Commun 2001;280:574–80. Lendeckel U, Kohl J, Arndt M, et al. Increased expression of ADAM family members in human breast cancer and breast cancer cell lines. J Cancer Res Clin Oncol 2005;131:41–8. Izumi Y, Hirata M, Hasuwa H, et al. A metalloproteasedisintegrin, MDC9/meltrin-gamma/ADAM9 and PKCdelta are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor. EMBO J 1998;17:7260–72. Roghani M, Becherer JD, Moss ML, et al. Metalloproteasedisintegrin MDC9: intracellular maturation and catalytic activity. J Biol Chem 1999;274:3531–40. Hotoda N, Koike H, Sasagawa N, Ishiura S. A secreted form of human ADAM9 has an alpha-secretase activity for APP. Biochem Biophys Res Commun 2002;293:800–5. Fischer OM, Hart S, Gschwind A, Prenzel N, Ullrich A. Oxidative and osmotic stress signaling in tumor cells is mediated by ADAM proteases and heparin-binding epidermal growth factor. Mol Cell Biol 2004;24:5172–83. Higashiyama S, Nanba D. ADAM-mediated ectodomain shedding of HB-EGF in receptor cross-talk. Biochim Biophys Acta 2005;1751:110–7. Hirao T, Nanba D, Tanaka M, et al. Overexpression of ADAM9 enhances growth factor-mediated recycling of E-cadherin in human colon cancer cell line HT29 cells. Exp Cell Res 2006;312:331–9. Tanida S, Joh T, Itoh K, et al. The mechanism of cleavage of EGFR ligands induced by inflammatory cytokines in gastric cancer cells. Gastroenterology 2004;127:559–69. Shintani Y, Higashiyama S, Ohta M, et al. Overexpression of ADAM9 in non-small cell lung cancer correlates with brain metastasis. Cancer Res 2004;64:4190–6. Mahimkar RM, Visaya O, Pollock AS, Lovett DH. The disintegrin domain of ADAM9: a ligand for multiple beta1 renal integrins. Biochem J 2005;385:461–8. Kristiansen G, Pilarsky C, Wissmann C, et al. Expression profiling of microdissected matched prostate cancer samples reveals CD166/MEMD and CD24 as new prognostic markers for patient survival. J Pathol 2005;205:359–76.

Editorial Comment on: ADAM9 Expression is a Significant and Independent Prognostic Marker of PSA Relapse in Prostate Cancer Alessandra Valentini, Sergio Bernardini Department of Internal Medicine, University of Rome ‘‘Tor Vergata’’, Rome, Italy [email protected]

[16] Ohl F, Jung M, Xu C, et al. Gene expression studies in prostate cancer tissue: which reference gene should be selected for normalization? J Mol Med 2005;83:1014–24. [17] Grutzmann R, Luttges J, Sipos B, et al. ADAM9 expression in pancreatic cancer is associated with tumour type and is a prognostic factor in ductal adenocarcinoma. Br J Cancer 2004;90:1053–8. [18] Karan D, Lin FC, Bryan M, et al. Expression of ADAMs (a disintegrin and metalloproteases) and TIMP-3 (tissue inhibitor of metalloproteinase-3) in human prostatic adenocarcinomas. Int J Oncol 2003;23:1365–71. [19] O’Shea C, McKie N, Buggy Y, et al. Expression of ADAM-9 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. [22] Carl-McGrath S, Lendeckel U, Ebert M, Roessner A, Rocken C. The disintegrin-metalloproteinases ADAM9, ADAM12, and ADAM15 are upregulated in gastric cancer. Int J Oncol 2005;26:17–24. [23] Le Pabic H, Bonnier D, Wewer UM, et al. ADAM12 in human liver cancers: TGF-beta-regulated expression in stellate cells is associated with matrix remodeling. Hepatology 2003;37:1056–66. [24] Grutzmann R, Foerder M, Alldinger I, et al. Gene expression profiles of microdissected pancreatic ductal adenocarcinoma. Virchows Arch 2003;443:508–17. [25] Alldinger I, Dittert D, Peiper M, et al. Gene expression analysis of pancreatic cell lines reveals genes overexpressed in pancreatic cancer. Pancreatology 2005;5:370–9. [26] Chin K, DeVries S, Fridlyand J, et al. Genomic and transcriptional aberrations linked to breast cancer pathophysiologies. Cancer Cell 2006;10:529–41. [27] Russ AP, Lampel S. The druggable genome: an update. Drug Discov Today 2005;10:1607–10. [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 tumorstromal interactions. Cancer Res 2005;65:4728–38. [30] Shigemura K, Sung SY, Kubo H, et al. Reactive oxygen species mediate androgen receptor- and serum starvation-elicited downstream signaling of ADAM9 expression in human prostate cancer cells. Prostate 2007;67:722–31.

Epidermal growth factor, insulin-like growth factor, and mutations of androgen receptor play a role in the progression of hormone-sensitive prostate cancer to hormone insensitivity. Although hormonal therapy is used earlier in the course of disease and a primary response rate of 80–90% occurs with hormonal ablation, almost all

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patients advance to a state of androgen independence manifested by increasing prostate-specific antigen (PSA) levels and new lesions on bone scans. The median survival time in patients with androgen-independent prostate cancer is about 18 mo and this condition is also accompanied by the attainment of high resistance to cytotoxic drugs [1]. Androgen independence may be an intrinsic, but resting, property of some prostate cancer cells that are activated in response to androgen deprivation. Only epithelial cells, not basal or stromal cells, undergo apoptosis on androgen withdrawal. The clonal selection hypothesis relates to the selective survival of preexisting androgen-independent cells within the tumour by hormonal deprivation [2]. These clonal cells are thought to be poorly differentiated and similar to basal or stem cells [3]. Substantial attempts have been expended in describing the available factors and determining their predictive value for staging, cancer recurrence, survival, and androgen-independent development. Interesting observations are constantly being made concerning the expression of a wide range of cellular components, including enzymes, structural proteins, ion channels, ligand receptors, gene sequences, among others, that are associated with progression of prostate cancer. Gene products of the A disintegrin and metalloproteinase (ADAM) family are critically involved in carcinogenesis and tumour progression of various solid tumours. It has been described that prostate tumours revealed high levels of ADAM9 mRNA in well-differentiated carcinomas, but only low or undetectable levels in poorly differentiated carcinomas. The loss-of-function experiments suggest that ADAM9 is critical for tumour progression past the well-differentiated state, whereas gain-offunction experiments provided additional evidence for a causal role of ADAM9 in tumorigenesis. ADAM9 deficiency delays or prevents prostate tumour progression to advanced states in a mouse prostate cancer model, whereas deregulated expression of ADAM9 initially disrupts epithelial

stromal homeostasis in the prostate epithelium, and later leads to prostatic intraepithelial neoplasia lesions [4]. Interestingly, Fritzsche et al found that ADAM9 was significantly up-regulated in prostate cancer in comparison to normal tissue on mRNA and protein levels. ADAM9 protein expression was significantly associated with shortened PSA relapse-free survival in univariate and multivariate analyses, particularly in patients who had received prior androgen ablation [5]. Additionally, the authors hypothesise that ADAM9 is a predictive marker of response to antiandrogenic therapy. Thus, this last hypothesis seems to be more interesting than the single value of ADAM9 as an independent prognostic factor, to individualise the gene as a molecular marker for hormone-independent switching of prostate cancer.

Editorial Comment on: ADAM9 Expression is a Significant and Independent Prognostic Marker of PSA Relapse in Prostate Cancer Colin Cooper Institute of Cancer Research, Sutton, UK [email protected]

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

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 cancer to androgen ablation therapy as studied in the Dunning R-3327-H adenocarcinoma. Cancer Res 1981;41: 5070–5. [3] Rashid MH, Chaudhary UB. Intermittent androgen deprivation therapy for prostate cancer. Oncologist 2004;9: 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, To¨lle 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

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ADAM9 and other members of this family are responsible for catalyzing their release from the cell by ectodermal shedding, a process vital for cellto-cell communication and for cellular homeostasis. ADAM9 is also a cell adhesion molecule that interacts with the integrin avb5 and that may be involved in controlling cell invasion. The elevated levels of this protein observed in many common neoplasms, including prostate, suggests a role for this protein in the development or progression of cancer, a view supported by the recent observation that the ADAM9 gene is both amplified and overexpressed in human breast cancer [1]. In the W10 mouse prostate cancer model knocking out the ADAM9 gene made the tumors that develop smaller and more differentiated in appearance, whereas in gain-of-function experiments overexpression of ADAM9 caused the development of prostatic intraepithelial hyperplasia [2]. In this issue Fritzsche et al [3] provide further compelling evidence for the importance of ADAM9 overexpression in determining aggression in human prostate cancer. In a study of 198 prostatectomy cases they showed that ADAM9 expression was significantly associated with shortened disease-free relapse. Importantly, in multivariate analyses the expression of ADAM9 provided prognostic information in addition to that obtained from stage, Gleason score, and preoperative prostate-specific antigen (PSA) level. This association still held when the authors considered only those patients treated with antiandrogen therapy prior to surgery. It has been shown in other studies that ADAM9 levels in prostate cancer cells

are controlled by androgens and by oxidative stress in such a way that induction of ADAM9 expression can be completely abrogated by the presence of antioxidants [4,5]. These results raise the prospect that the modulation of ADAM9 level or the development of drugs that directly target its catalytic activity may be of use in managing prostate cancer.

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, To¨lle 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 starvation-elicited downstream signaling of ADAM9 expression in human prostate cancer cells. Prostate 2007;67: 722–31. [5] Chin K, DeVries S, Fridlyand J, et al. Genomic and transcriptional aberrations linked to breast cancer pathophysiologies. 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