Med Oncol (2011) 28:475–480 DOI 10.1007/s12032-010-9481-8
ORIGINAL PAPER
ADAM-17 over-expression in gallbladder carcinoma correlates with poor prognosis of patients Kai Wu • Mingmei Liao • Bo Liu • Zhansheng Deng
Received: 17 February 2010 / Accepted: 2 March 2010 / Published online: 19 March 2010 Ó Springer Science+Business Media, LLC 2010
Abstract The ADAMs is a multi-functional gene family of membrane proteins possessing a disintegrin and metalloprotease domain. They have potential implications for the metastasis of human tumor cells via cell adhesion and protease activities. However, no studies have yet comprehensively examined the expression of ADAMs in gallbladder carcinoma. The aim of this study was to test the hypothesis that ADAM-17 (otherwise known as tumor necrosis factor-a converting enzyme) is involved in the progression of gallbladder carcinoma. Two hundreds samples of gallbladder carcinoma and sixty non-cancerous gallbladder samples were used to measure the expression of total ADAM-17 by enzyme-linked immunosorbent assay, and the precursor and active forms by western blotting analysis. Expression of ADAM-17 was significantly increased in tumors with high histological grade and pT stage compared with low histological grade and pT stage tumors and was not associated with patients’ gender, age, histological type, and resection margin involvement. Patients with high expression of ADAM-17 had a significantly shorter overall survival compared with those with low expression. Significantly, the prognostic impact of ADAM-17 was independent of conventional prognostic factors for gallbladder carcinoma. The current study demonstrated that the over-expression of ADAM-17 in patients with gallbladder carcinoma was linked closely with K. Wu M. Liao Z. Deng (&) The Xiangya Hospital, Central South University, 410011 Changsha, Hunan Province, China e-mail:
[email protected] B. Liu Department of General Surgery, The Second Xiangya Hospital, Central South University, 410011 Changsha, Hunan Province, China
histological grade, pT stage and prognosis, and thus provides further impetus for exploiting ADAM-17 as new target for the treatment of gallbladder carcinoma. Keywords Gallbladder carcinoma ADAM-17 Clinicopathology Prognosis
Introduction Gallbladder carcinoma is the most frequent biliary malignancy and a very lethal disease because of its early metastasis, strong invasion and poor prognosis. Only \5% patients with this disease may survive [1]. Standard treatment for gallbladder carcinoma consists of surgery and chemotherapy. Only 20–40% of patients with this disease can be performed curative resection, mainly because of the advanced disease stage at diagnosis [2]. Moreover, it has been demonstrated that gallbladder carcinoma is a chemotherapy-resistant tumor for several cytotoxic drugs [3]. Therefore, it is very important to find novel biomarkers for estimate the malignant degree and invasion tendency in order to guide the diagnosis of gallbladder carcinoma and to discover novel potent therapeutic strategies for treatment of this disease. The ADAMs is a multi-functional gene family of membrane proteins which possess a disintegrin and metalloprotease domain [4]. Some proteins of this family play a role in diverse biological processes such as fertilization, myogenesis, neurogenesis and the activation of growth factors [5]. The ADAMs have potential implications for the metastasis of human tumor cells in that they have the potential to regulate both extracellular matrix (ECM) remodeling and cell migration. Perhaps the best characterized ADAMs is ADAM-17 (otherwise known as
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tumor necrosis factor-a (TNF-a) converting enzyme, TACE), which was first identified through its ability to cleave the pro-inflammatory cytokine TNF-a from its membrane-bound precursor [6]. Other growth factors shown to be released by ADAM-17 include the epidermal growth factor-receptor (EGFR) ligands, transforming growth factor (TGF)-a, and amphiregulin, heparin-binding epidermal growth factor (HB-EGF), which may be related to tumor behavior [7]. Consistent with its ability to release these tumor-promoting factors, ADAM-17 has been implicated in tumor formation and progression. Recently, for example, ADAM-17-catalyzed shedding of TGF-a was found to be necessary for murine tumor formation, while in a three-dimensional culture of human breast cancer progression, inhibition of ADAM-17 reversed the malignant phenotype [8]. This reversion of the malignant phenotype resulted from the failure to release TGF-a and amphiregulin. McGowan et al. [9] also demonstrated that ADAM-17 may predict adverse outcome in patients with breast cancer. In addition, Takamune et al. [10] indicated that ADAM-17 expression was significantly correlated with the nodal metastasis and local recurrence in oral squamous cell carcinoma. However, no studies have yet comprehensively examined the expression of ADAM-17 in gallbladder carcinoma. The aim of this study was to test the hypothesis that ADAM-17 is involved in the progression of gallbladder carcinoma.
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chronic cholecystitis and/or cholelithiasis were also used as controls. Median patient follow-up at the time of analysis was 38 months. The end point investigated was overall survival (OS), defined as the time in months from operation to death from any cause. Measurement of ADAM-17 by enzyme-linked immunosorbent assay ADAM-17 protein levels in extracts from gallbladder carcinoma were measured by enzyme-linked immunosorbent assay (ELISA) (Quantikine, R&D Systems, MN, USA). The sensitivity of this assay was 0.6 ng/ml. ADAM-17 protein levels were measured according to the manufacturer’s instruction. Measurement of ADAM-17 by western blotting analysis ADAM-17 protein expression in extracts from gallbladder carcinoma was measured by western blotting analysis. Briefly, total protein was isolated from gallbladder tissue samples using 50 mM Tris–HCl (pH 7.4) containing Table 1 Clinicopathologic features of 200 patients with gallbladder carcinoma Factor
Materials and methods
The study was approved by the Research Ethics Committee of Xiangya Hospital, P. R. China. Informed consent was obtained from all of the patients. All specimens were handled and made anonymous according to the ethical and legal standards. Specimens from 200 patients with gallbladder carcinoma who consecutively underwent surgery between March 2004 and May 2009 in Xiangya Hospital were included in the present study. All specimens had been collected, diagnosed, and stored by the Department of Pathology, Xiangya Hospital. Samples were taken from 105 females and 95 males (aging 32–78 years, mean ± SD = 52.8 ± 10.6 years). Gallbladder carcinoma was diagnosed on the basis of histological findings and was staged according to the TNM system (nodal status and depth of wall infiltration) according to the American Joint Committee on Cancer. The clinicopathological features of the patients are summarized in Table 1. In addition, 60 non-cancerous gallbladder epithelium samples obtained from patients who underwent simple cholecystectomy for
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Percentage (%)
Gender Male
Patients and tissue samples
No.
Female Age at diagnosis
95
47.5
105
52.5
C70 years
132
66.0
\70 years
68
32.0
36
18.0
Histological grade I II
60
30.0
III
104
52.0
Histological type Adenocarcinoma
175
87.5
Squamous cell carcinoma
16
8.0
Neuroendocrine carcinoma
9
4.5
1
28
14.0
2
76
38.0
3 4
70 26
35.0 13.0
Negative
73
36.5
Positive
106
53.0
21
10.5
pT stage
Resection margin
Unknown
Med Oncol (2011) 28:475–480
protease inhibitors (2 ml/100 mg sample). Next, 100 lg of total protein was separated on 7% Tris–Glycine SDS gels. Following transfer onto nitrocellulose, the filter was blocked in 5% (wt/vol) milk (Marvel instant dried skimmed milk, Tesco, Ireland) in TBS, 0.05% Triton X-100 (vol/vol) (TBS-T) for 1 h at room temperature. Then, rabbit anti-human ADAM-17 polyclonal antibody (at the dilution of 1:100, ProSci Incorporated, Poway, CA) and mouse anti-human b-actin monoclonal antibody (at the dilution of 1:500, Santa Cruz, California, USA) were incubated with the membrane for 24 h at 4°C with endover-end rotation. Antibody–protein complexes were captured using 50% Gammabind G Sepharose (Pharmacia Biotech AB, Uppsala, Sweden), washed three times in PBS, and the Sepharose–antibody–protein complexes resuspended in 19 SDS–PAGE loading buffer. After that, samples were heated to 95°C for 10 min, resolved in 15% denaturing polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF; Immobilon, Millipore, MA) membranes. Finally, ADAM-17 was detected by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody and detection by enhanced chemiluminescence (PerkinElmer Life Sciences Inc., Boston, MA). Membranes were exposed to X-ray film (Fujifilm) in the dark for 15 min. Statistical analysis SPSS13.0 software for Windows (SPSS Inc, USA) and SAS 9.1 (SAS Institute, NC) was used for statistical analysis. Continuous variables were expressed as X s. Statistical analyses were performed with Fisher’s exact test for any 2 9 2 tables,Pearson v2 test for non-2 9 2 tables, Kaplan–Meier method for the question of survival, Chiquest trend test for ordinal datum, Cox regression analysis for the multivariate analysis. The p values of \0.05 were considered to be statistically significant.
Results Association between ADAM-17 protein levels determined by ELISA and the clinicopathological features of gallbladder carcinoma The mean protein level of ADAM-17 in gallbladder carcinomas was 3.88 ng/mg, which was significantly higher than that in non-cancerous gallbladder epitheliums (1.56 ng/mg, p = 0.01). The association of ADAM-17 protein levels determined by ELISA with the clinicopathological features of gallbladder carcinoma patients was shown in Table 2. Concentrations of ADAM-17 protein ranged from undetectable to 13.2 ng/mg with the median value of 3.88 ng/mg. We did not find any significant association of ADAM-17 protein levels
477 Table 2 Association between ADAM-17 protein levels and conventional clinicopathological parameters in 200 patients with gallbladder carcinoma Factor
Median ADAM-17 protein level (ng/mg)
pa
95
3.92
0.49
105
3.85
No.
Gender Male Female Age at diagnosis C70 years
132
3.97
\70 years
68
3.71
I
36
2.16
II
60
3.28
III
104
4.82
0.43
Histological grade 0.02
Histological type Adenocarcinoma
175
3.85
Squamous cell carcinoma
16
3.98
Neuroendocrine carcinoma pT stage
9
4.28
1
28
2.05
2
76
3.77
3
70
4.26
4
26
5.23
Negative
73
3.87
Positive
106
3.96
21
3.52
0.06
0.009
Resection margin
Unknownb a
V test
b
Excluded from p calculations
0.46
2
with patients’ gender and age, tumor histological type and resection margin involvement. However, the expression levels of ADAM-17 in the gallbladder carcinomas with high histological grade (4.82 ng/mg for grade III vs. 3.28 ng/mg and 2.16 ng/mg for grades II and I, respectively, p = 0.02) and pT stage (5.23 ng/mg for pT stage 4 vs. 4.26 ng/mg, 3.77 ng/mg and 2.05 ng/mg for pT stages 3, 2 and 1, respectively, p = 0.009) were significantly higher than those with low histological grades and pT stages. Correlation between ADAM-17 protein levels determined by ELISA and western blotting analysis All extracts from 200 patients with gallbladder carcinoma were also used for western blotting analysis. Consistent with the report of McGowan et al. [9], ADAM-17 protein also had two main forms in gallbladder carcinoma tissues, namely a 120-kDa precursor and a 100-kDa active form, following western blotting analysis. According to Spearman’s rank correlation analysis, we found that ADAM-17
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protein levels determined by ELISA were positively correlated with those by western blotting analysis in both the 120-kDa (p = 0.03, r = 0.37, n = 200) and 100-kDa form (p \ 0.01, r = 0.76, n = 200). Correlation between prognosis and clinicopathologic factors for patients with gallbladder carcinoma The prognosis of patients with high histological grade (III vs. II and I, p = 0.01), high pT stage (4 vs. 1 * 3, p = 0.008), positive resection margin (positive vs. negative, p = 0.02), and high ADAM-17 protein levels determined by ELISA (cut-off point was 25th percentile: p = 0.01; cut-off point was 50th percentile: p = 0.02; cutoff point was 75th percentile: p = 0.009) was significantly poorer than that of patients with low histological grade, low pT stage, negative resection margin, and low ADAM17 protein levels. However, the mean survival of gallbladder carcinomas was not associated with the patients’ gender, age, and histological type. Correlation between precursor and active forms of ADAM-17 and patients’ prognosis We also related both the precursor and active forms of ADAM-17 determined by western blotting analysis to patients’ prognosis. Using a cut-off at the 75th percentile value, patients with high levels of both the precursor (p = 0.01) and active (p = 0.008) forms had a significantly shorter OS than those with low levels (Fig. 1). Table 3 compared the prognostic utility of ADAM-17 expression with that of conventional prognostic factors for gallbladder carcinoma. As might be expected, tumor histological grade (p = 0.02), pT stage (p = 0.009), resection margin involvement (p = 0.03), and ADAM-17 (for precursor forms: p = 0.01; for precursor forms: p = 0.008) Fig. 1 Kaplan–Meier survival curves for ADAM-17 expression (‘a’ for 120 kDa— precursor form and ‘b’ for 100 kDa—active form) in gallbladder carcinoma using the 75th percentile as cut-off point. ADAM-17 was determined by western blotting. a, categorized by ADAM-17 protein expression \ the 75th percentile; b, categorized by ADAM-17 expression [ the 75th percentile
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significantly predicted poor outcome using univariate analysis. Significantly, in multivariate analysis, ADAM-17 correlated with shorter OS (for precursor forms: p = 0.02; for precursor forms: p = 0.009). Thus, the prognostic value of ADAM-17 was independent of the conventional prognostic factors mentioned above.
Discussion This is the first report demonstrating an independent prognostic value of ADAM-17 protein expression for gallbladder carcinoma. We found that the expression levels of ADAM-17 protein, as determined by ELISA, were independent of patients’ gender, age at diagnosis, tumor histological types and resection margin involvement. Significantly higher concentrations were, however, found in histological grade III compared with grade I and II tumors. Similarly, higher concentrations of ADAM-17 were found in those with pT stage 3–4 compared with pT stage 1–2, suggesting that ADAM-17 may play a role in the progression of gallbladder carcinoma. Consistent with the results of ELISA, neither the precursor nor the active forms of ADAM-17 were significantly related to patients’ gender, age at diagnosis, tumor histological types, and resection margin involvement. The expression of these two forms determined by western blotting analysis was positively correlated with the concentrations measured by ELISA. Additionally, using both ELISA and western blotting, we show here that elevated levels of ADAM-17 predict poor outcome in patients with gallbladder carcinoma independent of conventional prognostic factors for this disease, such as tumor histological grade, pT stage and resection margin involvement. Especially, the active form of ADAM-17 was a stronger predictor of outcome than the precursor form.
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Table 3 Univariate and multivariate analyses (Cox regression) of different prognostic parameters in patients with gallbladder carcinoma Factors
Univariate analysis
Multivariate analysis
P
Relative risk
95% confidence interval
P
Relative risk
95% confidence interval
ADAM-17 (120 kDa)a
0.01
2.13
1.98–3.73
0.02
1.69
1.33–3.26
ADAM-17 (100 kDa)a
0.008
2.68
1.08–5.97
0.009
3.82
1.02–5.61
Tumor grade pT stage
0.02 0.009
1.25 2.49
1.17–2.58 1.32–5.71
Not significant 0.02
1.16
1.2–3.91
Resection margin
0.03
1.21
1.16–3.52
0.04
1.09
0.98–3.27
a
Cut-off point for ADAM-17 was \75th versus [75th percentile
Gallbladder carcinoma has been associated with a dismal overall prognosis, and its incidence is still too high. Although the clinical course of gallbladder carcinoma has been thought to depend on the histological grades, pT stages of gallbladder carcinoma, as well as its resection margin involvement, are complex and not completely understood. So it is of great importance to be able to predict the biology of this disease and thus to predict its clinical course in the individual patient to ensure adequate treatment and patient monitoring. Molecular markers are being searched for and established to allow for a refined classification of prognosis, especially for the patient subgroups whose outcome can be only insufficiently predicted by the classical, previously established parameters [11, 12]. ADAMs is a gene family of multi-domain membraneanchored proteins, which comprise more than 30 members in various animal species and are implicated in several pathophysiological conditions [13]. ADAM-17 is the most widely investigated member in this family. It is reported to cleave a variety of substrates including HB-EGF, TGFa, TNF receptor, EGFR, c-fms, c-kit, p75NTR, growth hormone receptor, interleukin-6 receptor, interleukin-1 receptor, vascular cell adhesion molecule-1, CD40, bamyloid precursor, L-selectin, collagen VII, MUC1, and Notch [14–16]. The increased cleavage of such substrates as HB-EGF and TGFa by ADAM-17 is likely to be associated with tumor spread and growth because of their cell growth activity. In addition, ADAM-17 has also been demonstrated to be a part of VEGF-dependent, pro-angiogenic pathway leading to matrix metalloproteinases-2 activation and vessel formation [17]. There are several studies that have reported a correlation between ADAM-17 expression and tumor progression. In patients with colorectal carcinoma, high expression of ADAM-17 induces the activation of EGFR through the shedding of EGFR ligands [18]. In patients with ovarian cancer, ADAM-17 expression was significantly correlated with the expression of HB-EGF, which is increased in advanced ovarian cancer in comparison with that found in normal ovaries [19]. An increased expression of ADAM-17 has also been reported
to be linked to aggressive phenotypes of breast cancer [20] and prostate cancer [21]. Moreover, correlations between aberrant ADAM-17 expression and tumor outcome have been shown in some previous studies, which indicated that patients with oral, head, and neck squamous cell carcinoma showed correlation between ADAM-17 expression and prognosis [10]. Our results also indicated a significant correlation between ADAM-17 and prognosis of patients with gallbladder carcinoma. In this study, we used two different assays-ELISA and western blotting analysis to measure ADAM-17 expression patterns in gallbladder carcinoma. Each of these assays has specific advantages and disadvantages. Two methods were selected to complement each other. The main advantage of the ELISA is that it is quantitative. On the other hand, it measures total ADAM-17 and thus cannot separate the precursor and active fractions of the protein. Conversely, using western blotting, the precursor and active forms can be separated. This method, however, is only semiquantitative [9]. As the results, the consistent prognostic impact of the ELISA-determined concentrations with the precursor and active forms measured by western blotting was shown. Furthermore, we believe that an in silico approach or an immunohistochemical staining should be performed to validate the involvement of ADAM-17 in the progression of gallbladder carcinoma. According to the results of this study, ADAM-17 may serve as a new therapeutic target of gallbladder carcinoma for the following reasons. The first is that its elevated expression level occurs at an early clinical stage of gallbladder carcinoma. Second, ADAM-17 protein expression is strongly correlated with histological grade and pT stage of tumors. The third reason is ADAM-17 may predict the prognosis of patients with gallbladder carcinoma. In conclusion, the current study demonstrated that the over-expression of ADAM-17 in patients with gallbladder carcinoma was linked closely with histological grade, pT stage and prognosis and thus provides further impetus for exploiting ADAM-17 as new target for the treatment of gallbladder carcinoma.
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