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[Cancer Research 66, 9045-9053, September 15, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

Aberrant Expression of a Disintegrin and Metalloproteinase 17/Tumor Necrosis Factor-{alpha} Converting Enzyme Increases the Malignant Potential in Human Pancreatic Ductal Adenocarcinoma

Jörg Ringel1,2,3, Ralf Jesnowski1,2, Nicolas Moniaux4, Jutta Lüttges5, Jens Ringel2, Amit Choudhury4, Surinder K. Batra4, Günter Klöppel5 and Matthias Löhr1,2

1 Department of Medicine II, Mannheim Medical Faculty, University of Heidelberg; 2 Division of Molecular Gastroenterology, German Cancer Research Center, Heidelberg, Germany; 3 Department of Medicine A, University of Greifswald, Greifswald, Germany; 4 Department of Biochemistry and Molecular Biology and Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska; and 5 Department of Pathology, University of Kiel, Kiel, Germany

Requests for reprints: Matthias Löhr, Division of Molecular Gastroenterology (Deutsches Krebsforschungszentrum E180), German Cancer Research Center and Departments of Medicine II, Mannheim Medical Faculty, University of Heidelberg, Heidelberg, Germany. Phone: 49-621-383-2900; Fax: 49-621-383-1986; E-mail: matthias.loehr{at}med.ma.uni-heidelberg.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A disintegrin and metalloproteinase (ADAM) molecules are known for their unique potential to combine adhesion, proteolysis, and signaling. To understand the role of ADAM17/tumor necrosis factor-{alpha} (TNF-{alpha}) converting enzyme (TACE) in pancreatic ductal adenocarcinoma (PDAC), we investigated its expression, function, and in vitro regulation. ADAM17/TACE mRNA was expressed in 3 of 10 normal pancreatic tissues, 6 of 8 samples from patients with chronic pancreatitis, 10 of 10 PDAC tissues, and 9 of 9 pancreatic cancer cell lines, but it was absent in primary duct epithelial cells. Immunohistochemical staining revealed positive cancer cells in 8 of 10 PDACs but no staining of ducts in normal pancreas. ADAM17/TACE was found in 0 of 16 pancreatic intraepithelial neoplasia (PanIN)-1A lesions, 1 of 30 PanIN-1B lesions, 2 of 13 PanIN-2 lesions but, in 13 of 15 PanIN-3 lesions, associated with PDAC. Western blot, flow cytometry, and confocal microscopy analyses showed the aberrant expression of ADAM17/TACE protein in pancreatic cancer cell lines. The proteolytic activity of ADAM17/TACE, assessed by the release of TNF-{alpha}, was inhibited by TNF-{alpha} protease inhibitor. ADAM17/TACE gene silencing using small interfering RNA technique in vitro reduced invasion behavior dramatically, whereas proliferation was unaffected. Furthermore, ADAM17/TACE mRNA expression was down-regulated in pancreatic cancer cells arrested in G2-M phase as well as in a time-dependent manner after TNF-{alpha} and interleukin-6 incubation. In conclusion, our findings provide evidence of aberrant expression of the proteolytically active ADAM17/TACE in advanced precursor lesions (PanIN-3) and PDAC while identifying its critical involvement in the invasion process. (Cancer Res 2006; 66(18): 9045-53)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A gain in proteolytic capacity and alteration of the adhesiveness of cancer cells are of major importance during tumor invasion and progression. Although various roles of a large number of adhesion molecules and proteases with a broad spectrum of structural and functional characteristics have been investigated (1), the molecular basis of the highly aggressive behavior of pancreatic ductal adenocarcinoma (PDAC) remains unclear.

A disintegrin and metalloproteinase (ADAM) 17/tumor necrosis factor (TNF)-{alpha} converting enzyme (TACE) is a member of the new family of ADAM molecules (2). To date, at least 30 ADAMs have been identified in a variety of species (3). Like other members of this protein family, ADAM17/TACE is composed of a signal peptide, a propeptide, a metalloproteinase, a disintegrin, a cysteine-rich region, an epidermal growth factor (EGF)–like domain, a transmembrane region, and a cytoplasmic tail (2). ADAM molecules have the unique potential to combine various physiologic functions, including cell fusion, cell adhesion, intracellular signaling, and proteolytic processing of molecules (3–5). In addition, ADAM molecules play a critical role in malignant neoplasms (6, 7). A small subset of the presently known ADAM molecules show catalytic activity (8). ADAM17/TACE was originally described as being responsible for the proteolytic cleavage of the soluble form of TNF-{alpha}, which is located on the cell surface (2, 9). Subsequent studies have shown that ADAM17/TACE is also involved in the shedding of other biologically active proteins, including growth factors [erbB4/HER-4 and transforming growth factor (TGF)-{alpha}], surface molecules (L-selectin), and interleukin (IL) receptors (IL-R; IL-1R type II and IL-6R; refs. 10, 11). Any of these functions may play a role in a variety of diseases, including pancreatic cancer. Furthermore, ADAM17/TACE has been implicated in the processing of apoptosis- and survival-related factors, such as TNF receptors, p75 and p55, and TRANCE (12–14). Recently, the interaction of ADAM17/TACE and ADAM9 with cell cycle–related molecules raised the possibility of a link between ADAM proteins and cell cycle regulation (15).

Dysregulation of ADAM17/TACE has been shown in various diseases. However, there have been only a few reports about the role of ADAM molecules, especially ADAM17/TACE, in malignant tumors (16). Recent data have implicated TACE cleavage function in the activation of EGF receptor (EGFR) and EGFR signaling systems (17, 18), which regulate the proliferation and motility of squamous cell carcinoma cells in vitro (17). It has also been shown that ADAM17/TACE is overexpressed in mammary cancer (18).

Considering its potential functions, ADAM17/TACE might also be involved in the progression of pancreatic cancer. To date, no studies have been published that elucidate the expression, regulation, and function of ADAM17/TACE in pancreatic cancer.

In this study, we show the aberrant expression of proteolytic active ADAM17/TACE in pancreatic cancer cells and its involvement in invasion. Our immunohistochemical staining results, including the analyses of precursor lesions [pancreatic intraepithelial neoplasia (PanIN)], suggest ADAM17/TACE expression as a later event in PanIN progression to PDAC. Gene silencing experiments revealed the critical involvement of ADAM17/TACE in the invasion behavior of pancreatic cancer cells. Finally, our data provide insights into the in vitro regulatory mechanisms of ADAM17/TACE mRNA in pancreatic cancer cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. Polyclonal anti-human ADAM17/TACE (cytoplasmic domain), horseradish peroxidase (HRP)–conjugated secondary antibody, and goat anti-rabbit FITC-labeled antibodies were purchased from Calbiochem, Inc. (Temecula, CA). Nonspecific polyclonal rabbit control serum was obtained from Research Genetics, Inc. (Temecula, CA). Mouse anti-human ADAM17/TACE (extracellular domain) IgG1 and FITC-labeled mouse anti-human ADAM17/TACE IgG1 were obtained from R&D Systems (Minneapolis, MN); nonspecific FITC-labeled mouse IgG1 and CK19 antibodies were purchased from Sigma (St Louis, MO). The TNF-{alpha} ELISA kit was purchased from R&D Systems. The TNF-{alpha} protease inhibitor (TAPI) was generously provided by Immunotech Corp. (Seattle, WA).

Tissue specimens. Tissue samples (histologically proven) from normal human pancreas (n = 12), chronic pancreatitis patients (n = 10), and PDAC patients (n = 12) were obtained through an organ donor program and from surgical specimens from Inselspital Bern, Bern, Switzerland; Faculty Mannheim and Heidelberg, University of Heidelberg, Heidelberg, Germany (kindly provided by H. Fries); and University of Kiel, Kiel, Germany (J. Lüttges and G. Klöppel) in accordance with the stipulations of the local ethic committees.

Freshly removed tissue samples were fixed in 10% formaldehyde solution and paraffin embedded for histologic analysis. In addition, tissue samples were frozen in liquid nitrogen immediately after surgical removal and maintained at –80°C until RNA extraction.

Tissue samples were histologically proven and PanINs were classified using published criteria established at the National Cancer Institute–sponsored Pancreatic Cancer Think Tank (Park City, UT; ref. 19). The histologic features were graded by an experienced pancreatic pathologist (J.L.).

RNA isolation and reverse transcription-PCR. Total RNA from tissue samples and cell cultures and cDNA templates for reverse transcription-PCR (RT-PCR) were prepared as described previously (20, 21). The following intron-spanning primers were used:


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PCR conditions for ADAM17/TACE were as follows: initial denaturation for 5 minutes at 95°C, 35 cycles (95°C for 30 seconds, annealing at 55°C for 45 seconds, 30 seconds at 72°C), followed by 15 minutes at 72°C. For ADAM9, the conditions were used as published (22).

Negative controls omitting the reverse transcription reaction were included in the experiments. The density of DNA bands was determined using the GelExpert software system (Nucleotech Corp., Santa Mateo, CA). The density values for the ADAM17/TACE amplification products were normalized to those of GAPDH used as an internal control. Values are expressed as the mean ± SE.

To confirm the identity of the PCR products, the amplified DNA fragments were sequenced using vector-specific primers and an ABI PRISM sequencer (Applied Biosystems, Foster City, CA).

Immunohistochemistry. Following antigen demasking for 2 minutes with the pressure cooker method (23), 5-µm thin sections were routinely dewaxed and blocked with normal rabbit serum (diluted 1:20 in PBS) for 30 minutes. Antigens were detected using a standard three-step method with primary, biotinylated secondary antibody (1:50 working dilution) and streptavidin-HRP (1:300 in PBS). After blocking the endogenous peroxidase activity by 0.3% hydrogen peroxide in methanol and after two washes in PBS, the reaction was visualized with 3,3'-diaminobenzidine (DAKO, Hamburg, Germany).

Cell culture. The human pancreatic cancer cell lines BxPC-3, CAPAN-1, CAPAN-2, QGP1, T3M4, Colo357, PANC-1 [all from American Type Culture Collection (ATCC), Rockville, MD], and SW979 (gift from H. Kalthoff) and primary pancreatic fibroblasts (kind gift from P. Termuhlen, University of Nebraska Medical Center, Omaha NE) were cultured in DMEM with glutamax-I supplemented with 10% heat-inactivated FCS and antibiotics (100 units/mL penicillin and 100 µg/mL streptomycin-G, Invitrogen). Primary human pancreatic duct epithelial cells (PDEC) were prepared and cultured as described previously (24). The cells expressed the duct-specific cytokeratins 7 and 19, CA19-9, and carbonic anhydrase II. In addition, the human cervix carcinoma cell line HeLa (ATCC) was used. The human pancreatic tumor cell line HPAF/CD18 was derived from the heterogeneous HPAF pancreatic adenocarcinoma cell line (25). For the analysis of the regulation of ADAM17/TACE expression by growth factors and cytokines, PANC-1 cells were incubated with TGF-ß1 (5 and 10 pg/mL), TGF-ß2 (10 pg/mL), EGF (20 ng/mL), vascular endothelial growth factor (VEGF; 10 ng/mL; all from R&D Systems), IL-6 (10 ng/mL), IFN-{gamma} (10 ng/mL), TNF-{alpha} (10 ng/mL; all from PeproTech, Inc., Rocky Hill, NJ) for 12, 24, and 48 hours under serum-free conditions. Noncontact cocultivation of PANC-1 and primary fibroblasts was carried out using six-well plates and cell culture inserts (Becton Dickinson, Franklin Lakes, NY; ref. 21). After 12, 24, and 48 hours, cells were collected and RNA was prepared for subsequent analysis as described above. Controls were prepared by cultivation of fibroblasts and pancreatic cancer cells alone.

Preparation of cell lysates. In brief, after two washes with ice-cold PBS, cells were lysed by ice-cold radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 7.4), 1% NP40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, aprotinin, leupeptin, 1 mg/mL DTT]. After 30 minutes of incubation on ice, lysates were centrifuged at 1,000 x g for 10 minutes at 4°C. The protein concentration of the lysates was determined using the Bio-Rad detergent-compatible protein assay (Bio-Rad, Hercules, CA).

Immunoblotting. Equal amounts of cell lysates were resolved by SDS-PAGE and blotted to polyvinylidene difluoride membranes (Amersham Pharmacia, Little Chalfont, England). The membranes were blocked in 5% dry milk in TBST-Tween 20 [TBST; 50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.05% Tween 20] for 1 hour at room temperature and incubated overnight at 4°C with the polyclonal anti-human ADAM17/TACE antibody (Chemicon, Temecula, CA) diluted at 1:500 in TBST containing 3% dry milk. After repeated washes, the HRP-conjugated secondary antibody (Chemicon), diluted at 1:2,000 in 3% dry milk in TBST, was added and incubated at room temperature for 1 hour. After additional washes, enhanced chemiluminescence (ECL) reagents (Amersham, Arlington Heights, IL) were applied, and blots were exposed to ECL-sensitive film (Kodak, Rochester, NY). Anti-CK19 (Sigma) antibody was used as control for equal loading.

Flow cytometry. For the analysis of ADAM17/TACE surface expression, cells were gently detached using 1 mmol/L EDTA solution and washed with PBS thrice (supplemented with 0.5% bovine serum albumin). Cells (5 x 105) were incubated with fluorescein-conjugated mouse anti-human TACE monoclonal antibody (mAb) or FITC-labeled isotype-matched mouse control mAb for 30 minutes at 4°C. The cells were then washed once with PBS. ADAM17/TACE surface expression was analyzed by measuring 10,000 cells from each sample in a FACScan flow cytometer (Becton Dickinson, Heidelberg, Germany) using CellQuest software (Becton Dickinson, Heidelberg, Germany).

Confocal microscopy. To examine the expression of endogenous TACE, pancreatic cancer cells seeded onto glass coverslips were fixed and permeabilized with 100% methanol for 2 hours at –20°C and acetone for 15 minutes. After blocking with 5% normal rabbit serum, the fixed cells were incubated with the polyclonal rabbit anti-TACE antibody raised against the cytoplasmic tail (1:100 dilution) or in nonspecific rabbit serum diluted in PBS for 2 hours at room temperature. The coverslips were washed six times and the secondary FITC-labeled antibody (Sigma) was incubated for 30 minutes at room temperature. After an additional washing step, the glass coverslips were mounted onto glass slides. Cells were analyzed using a LSM 410 Zeiss Confocal Microscope (Zeiss, Wetzlar, Germany).

TNF-{alpha} ELISA and ADAM17/TACE inhibition assay. The proteolytic activity of ADAM17/TACE was assessed by measuring the release of TNF-{alpha}, which is the main target molecule of the ADAM17/TACE protease activity. To evaluate the TNF-{alpha} concentration in the medium, PANC-1 cells were grown in DMEM supplemented with 10% FCS for 24 hours. After repeated washing with PBS, cells were incubated with DMEM without FCS for 2 hours. Various concentrations of the TACE-inhibitor TAPI (100, 50, and 1 µmol/L; Immunex Corp., Seattle, WA) were added to the medium for 2 hours to inhibit the TNF-{alpha} shedding function of ADAM17/TACE.

After incubation, all supernatants were collected and centrifuged. The number of cells was determined. The TNF-{alpha} concentration in the cell medium was measured by ELISA (R&D Systems) as recommended by the manufacturer. The experiments were analyzed in ELISA measurements in triplicate.

RNA interference. Because all investigated pancreatic cancer cell lines express ADAM17/TACE, its function was investigated by ADAM17/TACE gene silencing induced by the introduction of small interfering RNA (siRNA) duplexes in different pancreatic cancer cell lines. Transfection of 21 nucleotide siRNA duplexes (Qiagen) was carried out using RNAiFect reagent (Qiagen). According to published data (17), a mixture of three siRNA duplexes targeting different regions of the ADAM17/TACE gene was used for high efficient silencing. Nonsilencing control siRNA was used as control for nonspecific silencing effects (RNA interference control kit, Qiagen). Transfection conditions, including amount of siRNA and transfectant reagent (RNAiFect), were optimized using Alexa Flour 488–labeled siRNA (Qiagen). The highest efficiency of gene silencing in CAPAN-1 cells was obtained by using 1.5 µg siRNA mixture. Specific silencing of the ADAM17/TACE gene was confirmed by RT-PCR 3 and 4 days after transfection and by Western blot 4, 5, and 6 days after transfection as described above.

Proliferation and invasion assays. For proliferation analysis, ADAM17/TACE gene silenced CAPAN-1 cells (6 days after siRNA transfection) as well as cells with the control siRNA were seeded onto a 96-well dish (3,000 cells per well) and grown with DMEM supplemented with 10% FCS for 3 days. Proliferation was analyzed with the water-soluble tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate; Roche) according to the manufacturer's instructions using an ELISA reader 2 hours after WST-1 addition. Invasion behavior was assayed using Matrigel-coated Transwell systems (21). In brief, 1 x 104 ADAM17/TACE gene–silenced CAPAN-1 cells as well as cells with the control siRNA were seeded into Matrigel-coated 24-well Transwell inserts (8.0-µm pore size, Costar Transwell) in DMEM with 10% FCS. After 72 hours of incubation, the Transwells were removed from the plates. The cells that invaded through the filter membrane were fixed with 100% ethanol and stained with 0.4% trypan blue. The noninvaded cells on top of the Transwell were scraped with a cotton swab. The invaded cells in three high power fields of each sample were counted under a light microscope.

Cell cycle synchronization. Cell cycle synchronization was done as described by Pedrali-Noy et al. (26). In brief, PANC-1 cells were arrested with 5 mg/mL aphidicolin (Sigma) for 24 hours and then released in fresh complete medium. Four and 9 hours after release, the cells were trypsinized and processed for RNA preparation and cell cycle analysis. The concentration and exposure time for aphidicolin were optimized in preliminary studies.

The cell cycle phases were measured by flow cytometry using DNA staining with propidium iodide as described previously (27). The cell cycle analysis was done using a FACStar flow cytometer (Becton Dickinson, Franklin Lakes, CA).

Statistics. Results are expressed as mean ± SE of at least three separate experiments. For statistical analysis, Student's t test was used. P < 0.05 was considered to be statistically significant.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ADAM17/TACE expression in normal pancreas, chronic pancreatitis, and PDAC. First, the expression of ADAM17/TACE mRNA was analyzed in a panel of tissue samples using RT-PCR. The ADAM17/TACE amplification product of the expected size of 434 bp was only detectable in 3 of 10 normal pancreas samples but in 10 of 10 invasive PDAC tissues and in 6 of 8 chronic pancreatitis tissue samples (Fig. 1A ).


Figure 1
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  		Figure 1. ADAM17/TACE mRNA and protein expression in human pancreatic tissues (A and B) and in human pancreatic cancer cell lines (C and D). A, ADAM17/TACE mRNA expression analyzed by RT-PCR in normal pancreas (1), chronic pancreatitis samples (2 and 3), ductal pancreatic adenocarcinoma tissues (4-7), the cervix carcinoma cell line HeLa as positive control (8), and the negative control without cDNA (9). The housekeeping gene GAPDH (924 bp) was amplified as internal control. B, ADAM17/TACE immunostaining of specimens of normal pancreas (a), chronic pancreatitis (b), and ductal adenocarcinoma (c and d) and of ductal adenocarcinoma–associated PanIN lesions (e and f). a, in normal pancreas tissue, ADAM17/TACE immunoreactivity was absent in ductal, acinar, and islet cells; b, similarly, the ductal cells in the chronic pancreatitis sample were negative; c and d, ductal adenocarcinoma with a predominantly membrane-bound immunolabeling for ADAM17/TACE. Original magnification, x100. e, PanIN-1A lesions are composed of tall columnar cells. None of the PanIN-1A lesions displayed a ADAM17/TACE staining; f, an example of a PanIN-3 lesion that harbored membrane-bound ADAM17/TACE. Original magnification, x200. C, RT-PCR of ADAM17/TACE mRNA (top) in pancreatic fibroblasts as negative control (1), PDEC (2), CAPAN-1 (3), CAPAN-2 (4), BxPC-3 (5), PANC-1 (6), HPAF/CD18 (7), T3N4 (8), QGP1 (9), SW979 (10), Colo357 (11), the cervix carcinoma cell line HeLa as positive control (12), and the negative control without cDNA (13). Bottom, housekeeping gene GAPDH as internal control. M, 100-bp DNA molecular weight marker. Right, sizes of the amplification products. D, Western blot analysis of ADAM17/TACE expression in the pancreatic cancer cell lines: (1) CAPAN-1, (2) CAPAN-2, (3) BxPC-3, (4) HPAF/CD18, (5) PANC-1, (6) T3N4, (7) QGP1, (8) SW979, (9) the cervix carcinoma cell line HeLa as positive control, (10) and fibroblast as negative control. Margins, positions of the protein molecular weight markers. The membrane was analyzed with a polyclonal rabbit anti-human ADAM17/TACE serum (Chemicon) raised against the cytoplasmic part of ADAM17/TACE as described.

 
In normal pancreas tissue, ADAM17/TACE immunoreactivity was absent in ductal, acinar, and islet cells (Fig. 1B, a). Similarly, the ductal cells in the chronic pancreatitis specimens were negative (Fig. 1B, b), except for a few ductal structures surrounded by positive inflammatory cells in one chronic pancreatitis sample (Table 1 ).


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  		Table 1. Immunostaining of ADAM17/TACE expression in PanINs from chronic pancreatitis and PDAC tissue samples

 
In contrast, moderate to strong membrane-bound ADAM17/TACE immunoreactivity was present in 10 of 12 PDACs, irrespective of the tumor grade (Fig. 1B, c and d; Table 1). Faint cytoplasmic staining was observed in a few centroacinar and neuroendocrine cells in one specimen (data not shown). To study the ADAM17/TACE expression during the development of the human PDACs, we used PanIN lesions associated with the PDACs. A total of 53 PanIN lesions were found in 9 of 12 investigated specimens of PDACs. ADAM17/TACE was found in 0 of 16 PanIN-1A lesions, 1 of 16 PanIN-1B lesions, and 2 of 6 PanIN-2 lesions (Fig. 1B, e; Table 1). However, 13 of 15 of the PDAC-associated high-grade PanIN-3 precursor lesions were found to harbor membrane-bound ADAM17/TACE protein (Fig. 1B, f; Table 1). In the chronic pancreatitis specimens, 14 PanIN-1B and 7 PanIN-2 lesions were identified. None of these chronic pancreatitis–associated PanINs showed ADAM17/TACE staining (Table 1).

Expression and localization of ADAM17/TACE expression in human pancreatic cancer cell lines. Using RT-PCR, ADAM17/TACE was not detectable in PDEC or pancreatic fibroblasts (Fig. 1C). In contrast, all investigated pancreatic cancer cell lines showed the expected amplification product of 434 bp, which was confirmed by sequencing. The amplification of the housekeeping gene GAPDH showed the integrity of the used mRNA (Fig. 1C).

Expression of ADAM17/TACE on the protein level was confirmed in the pancreatic cancer cell lines by various methods. Western blot analysis revealed two immunoreactive bands with apparent molecular masses of 120 and 100 kDa under reducing conditions using the polyclonal antibody specific for the cytoplasmic portion of ADAM17/TACE. The 120-kDa band, described previously as the TACE precursor form, was present in seven of eight pancreatic cancer cell lines and in the control cell line HeLa (Fig. 1D). The second immunoreactive band with a molecular mass of 100 kDa probably represents the mature form of TACE/ADAM17. This band was fainter compared with the precursor form in the pancreatic cancer cell lines and in the positive control (Fig. 1D). In the pancreatic cancer cell line CAPAN-2, only the 120-kDa band was present, whereas, in the T3N4 cell line, only the 100-kDa band was detectable (Fig. 1D). Furthermore, a few samples displayed an additional immunoreactive band with an approximate molecular weight of 20 kDa (Fig. 1D).

Flow cytometrical analysis using an antibody detecting the extracellular domain of ADAM17/TACE revealed that ADAM17/TACE protein was expressed on the cell surface in all investigated pancreatic cancer cell lines with very similar densities (Fig. 2A ). ADAM17/TACE protein was not detectable in pancreatic ductal cells (Fig. 2A).


Figure 2
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  		Figure 2. Cellular localization of ADAM17/TACE in pancreatic cancer cells. A, fluorescence-activated cell sorting (FACS) histograms showing the surface expression of ADAM17/TACE in pancreatic cancer cell lines, PDEC, and HeLa cells. The cells were stained with FITC-labeled mouse anti-human ADAM17/TACE IgG1 (open histograms) and the FITC-labeled isotype-matched control antibody (shaded histograms). Cells were as follows: (a) CAPAN-1, (b) PANC-1, (c) BxPC-3, (d) PDEC, and (e) HeLa as control. Representative FACS analyses. B, intracellular localization of ADAM17/TACE in CAPAN-1 (a) and PANC-1 (c) cells using immunofluorescence staining. The cells were incubated with either anti-ADAM17/TACE cytoplasmic tail rabbit serum (a and c) or nonspecific rabbit control serum (b and d) followed by FITC-labeled goat anti-rabbit secondary antibody. CAPAN-1 (a) and PANC-1 (c) cells showed a specific diffuse cytoplasmic staining pattern (original magnification, x100) with more intense staining in the perinuclear region. Only low levels of fluorescence staining were detected when cells were stained with the nonspecific rabbit control serum (b and d). Representative of the results observed in three experiments.

 
In addition to the cell surface expression of the ADAM17/TACE protein, studies have shown that ADAM17/TACE is present in the cytoplasm of some cell types (28). Confocal microscopy in permeabilized pancreatic cancer cells revealed a diffuse cytoplasmic staining pattern with more intense staining in the perinuclear region (Fig. 2B, a and c). Control cells treated with nonimmune rabbit serum or with the secondary antibody alone showed insignificant immunostaining (Fig. 2B, b and d).

Inhibition of TNF-{alpha} shedding by TAPI. The proteolytic activity of ADAM17/TACE was assessed by measuring the release of TNF-{alpha}. For this purpose, the concentration of TNF-{alpha} in the supernatant of PANC-1 cell cultures was measured in the presence or absence of different concentrations of TAPI using the ELISA technique. This inhibitor has been reported to effectively block the release of TNF-{alpha} from cells (2). Consistent with previous studies about the endogenous expression in pancreatic cancer cell lines, TNF-{alpha} was detectable in the supernatant of PANC-1 cells after 2 hours of culture in FCS-free medium (Fig. 3A ). Inhibition of the protease activity of ADAM17/TACE using the specific inhibitor TAPI dose dependently decreased the release of TNF-{alpha} (Fig. 3A).


Figure 3
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  		Figure 3. Effects of the TAPI on the release of soluble TNF-{alpha} (A) and of ADAM17/TACE gene silencing on invasion and proliferation behavior (B-D). A, for measurement of the TNF-{alpha} concentration in the medium, PANC-1 cells were incubated with DMEM without FCS and different concentrations of TAPI for 2 hours. The TNF-{alpha} concentration in the supernatants was measured using the ELISA system as recommended by the manufacturer. Columns, mean (n = 3 independent experiments); bars, SE. P < 0.001, t test. ADAM17/TACE gene silencing was done by siRNA transfection in CAPAN-1 cells. RNA was extracted 3 days after siRNA transfection and RT-PCR was done as described above. B, top, ADAM17/TACE; bottom, GAPDH. 1, CAPAN-1 with control siRNA; 2, CAPAN-1 with ADAM17/TACE siRNA; 3, CAPAN-1. M, molecular weight marker. C, top, Western blot analyses of ADAM17/TACE expression. 1, CAPAN-1 control; 2, CAPAN-1 4 days after ADAM17/TACE siRNA transfection; 3, CAPAN-1 5 days after ADAM17/TACE siRNA transfection; 4, CAPAN-1 6 days after ADAM17/TACE siRNA transfection. Bottom, the immunoreactive band of CK19 (40 kDa) as control. Margins, positions of the molecular weight markers. D, top, proliferation of ADAM17/TACE-silenced CAPAN-1 cells as well as cells transfected with control siRNA was measured after WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) incubation by ELISA reader as described; bottom, invasion behavior was assayed using Matrigel-coated Transwell systems. The cells invaded through the filter membrane were counted in three high power fields of each sample under a light microscope after fixation and staining as described in Materials and Methods.

 
ADAM17/TACE gene silencing, proliferation, and invasion. To verify the effect of ADAM17/TACE on proliferation and invasion, PANC-1 and CAPAN-1 cells were transfected with siRNA to block the endogenous expression of ADAM17/TACE. Suppression of ADAM17/TACE was monitored by RT-PCR and Western blot analyses (Fig. 3B and C). In CAPAN-1 cells, ADAM17/TACE mRNA was undetectable 3 days after siRNA transfection (Fig. 3B). At protein level, ADAM17/TACE was almost suppressed on day 6 after siRNA transfection (Fig. 3C). In contrast, despite the use of various transfection conditions, there was no suppression of ADAM17/TACE at the mRNA or protein level in PANC-1 cells (data not shown).

Therefore, ADAM17/TACE and control siRNA–transfected CAPAN-1 cells were used for further proliferation and invasion experiments. In CAPAN-1 cells transfected with siRNAs against ADAM17/TACE, the number of invading cells was reduced to 1.9% of control siRNA–transfected cells (Fig. 3D, bottom). Proliferation assays revealed no significant difference in proliferation of CAPAN-1 cells after transfection of siRNA against ADAM17/TACE compared with the control cells (Fig. 3D, top).

ADAM17/TACE mRNA expression is cell cycle dependent. We used a aphidicolin-mediated S-phase block to synchronize PANC-1 cells. When aphidicolin was removed after 24 hours, PANC-1 cells were blocked at G1-S phase (85-92%) as shown in Fig. 4A . Nine hours after release in fresh medium, 77% to 85% of the cells were in the G2-M phase, whereas the percentage of cells at the G1-S phase decreased dramatically (10-22%; Fig. 4B). In contrast, in untreated PANC-1 cells, the percentage of cells in the G1-S phase ranged from 60% to 70% and in G2-M phase from 30% to 40% of the cell population (data not shown).


Figure 4
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  		Figure 4. Cell cycle phase–dependent expression of ADAM17/TACE mRNA. PANC-1 cells were arrested at different cell cycle phases using aphidicolin; RNA was extracted and RT-PCR was done as described in Materials and Methods. The cell cycle phases were determined using propidium iodide staining and FACS analysis. A, representative DNA histogram of G1-S-arrested PANC-1 cells. Cells (85%) are arrested in the G1-S phase and 15% in the G2-M phase. B, nine hours after aphidicolin treatment, 77% of the cells are arrested in the G2-M cell cycle phase (23% G1-S). C, 1, asynchronous PANC-1 cells; 2, G2-M phase–arrested cells; 3, G1-S phase–arrested cells; 4, negative control. Top, expression of ADAM9 transcript; bottom, expression of ADAM17/TACE mRNA. Right, molecular sizes. D, relative quantification of the ADAM9 and ADAM17/TACE PCR products. The density values for the ADAM17/TACE transcripts (434 bp) and ADAM9 transcripts (390 bp) were normalized against those of the coamplified GAPDH (924 bp). Columns, mean (n = 5); bars, SE.

 
Corresponding to the different cell cycle phases, we found significant alterations in ADAM17/TACE mRNA expression, whereas GAPDH was almost unaffected (Fig. 4C, bottom). In PANC-1 cells blocked at the G2-M phase, the level of ADAM17/TACE mRNA was dramatically (~60-fold) decreased compared with cells arrested in G1-S phase (Fig. 4C, bottom and Fig. 4D). To determine whether this regulation profile is unique to ADAM17/TACE, we investigated the expression of ADAM9, which is also known to interact with a cell cycle checkpoint-associated protein (15). ADAM9 mRNA was undetectable in PANC-1 cells arrested in G2-M phase, whereas it was present in untreated cells and in cells arrested in G1-S phase (Fig. 4C, top and Fig. 4D).

Regulation of ADAM17/TACE mRNA expression by external factors. To assess further regulatory mechanisms of ADAM17/TACE expression in pancreatic cancer cells, we treated PANC-1 cells with growth factors and cytokines known to play a role in pancreatic cancer and/or to be associated with the function of ADAM17/TACE.

First, we tested whether serum may influence the ADAM17/TACE mRNA expression. Semiquantitative RT-PCR revealed an ~3-fold decrease of mRNA in PANC-1 cells under serum-free culture conditions (Fig. 5A and B ). Similar results were seen in other pancreatic cancer cell lines too (data not shown). In contrast to endothelial cells, the treatment with TNF-{alpha} for 12 hours reduced the ADAM17/TACE mRNA expression nearly completely (Fig. 5A and B). This effect was also detectable after 24 hours of incubation, whereas after 48 hours, the expression level returned to the starting point of the untreated cells (Fig. 5A and B). Similarly, IL-6 incubation decreased in a time-dependent manner ADAM17/TACE mRNA expression (Fig. 5A and B). However, other factors, including TGF-ß1, TGF-ß2, EGF, VEGF, and IFN-{gamma} did not alter ADAM17/TACE mRNA expression (data not shown).


Figure 5
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  		Figure 5. Analysis of ADAM17/TACE mRNA expression after incubation of PANC-1 cells with TNF-{alpha} and IL-6 using semiquantitative RT-PCR. The cells were grown in the presence (1) or absence (2) of 10% bovine calf serum for 3 days. The incubation with TNF-{alpha} and IL-6 was done as described in Materials and Methods. A, representative RT-PCR result. GAPDH was used as internal PCR control. 1, PANC-1 cells with 10% FCS; 2, PANC-1 cells without FCS; 3, 10 ng/mL TNF-{alpha} for 12 hours; 4, 10 ng/mL TNF-{alpha} for 24 hours; 5, 10 ng/mL IL-6 for 12 hours; 6, 10 ng/mL IL-6 for 24 hours; 7, 10 ng/mL TNF-{alpha} for 48 hours; 8, 10 ng/mL IL-6 48 hours. M, molecular weight marker. B, relative quantification of the ADAM17/TACE PCR product. The density values for the ADAM17/TACE transcripts (434 bp) were normalized against those of the coamplified GAPDH (924 bp). Columns, mean; bars, SE.

 
In addition, cocultivation of PANC-1 and primary pancreatic fibroblasts did not alter the expression of ADAM17/TACE mRNA in the pancreatic cancer cells (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ADAM molecules, with their unique potential to combine adhesion, proteolysis, and signaling, are involved in a variety of cellular functions, including processing of proteins, interactions with integrin receptors, and signaling molecules. Our data provide new insights into the expression, function, and developmental implications of the disintegrin-metalloproteinase ADAM17/TACE, which was detected on the surface and in the cytoplasm of PDAC cells. In addition, our data indicate that the ADAM17/TACE regulatory mechanism is cell cycle dependent.

Expression of ADAM17/TACE has been detected in various hematopoietic cancer cell lines, in prostate cancer cell lines, and in squamous cell carcinoma cells (17, 22, 29). These studies did not include normal or preneoplastic tissues to determine the possible role of ADAM17/TACE in the development of malignancy.

Our investigations revealed aberrant expression of ADAM17/TACE in PDAC. ADAM17/TACE mRNA was detectable in only a few normal tissue samples, whereas it was present in all human pancreatic cancer tissues. In addition, ADAM17/TACE was detectable in pancreatic cancer cell lines but not in human primary ductal epithelial cells. Because the current model of PDAC is based on the concept of a stepwise progression from normal ductal epithelium over increasing grades of PanIN to pancreatic adenocarcinoma, we studied the timing of ADAM17/TACE expression in the development of human PDAC. Whereas not a single PanIN-1A lesion showed ADAM17/TACE staining, 86% of the high grade PanIN-3 lesions expressed ADAM17/TACE. Chronic pancreatitis as a benign disease is associated with an increased risk of developing pancreatic cancer. Thus, we compared ADAM17/TACE expression in chronic pancreatitis and PDAC samples. Similar to PDAC samples, ADAM17/TACE mRNA was detected in most chronic pancreatitis cases. But, in contrast to PDACs, immunostaining was only positive in one specimen in duct areas with surrounding inflammatory cells. Moreover, there was no detectable staining in 21 chronic pancreatitis–associated PanIN lesions. Thus, ADAM17/TACE might be useful as a diagnostic marker of pancreatic cancer to distinguish between PDAC and chronic pancreatitis. Altogether, these findings suggest that ADAM17/TACE expression is a late event in the development of PanINs and progression to PDAC.

To assess the expression, proteolytic activity and biological function, and possible regulatory mechanisms of ADAM17/TACE in pancreatic cancer, we analyzed established PDAC cell lines, including CAPAN-1 and PANC-1. Our data on TACE protein expression in pancreatic cancer cell lines and PDEC correlated with the immunohistochemistry data. In addition to the immunoreactive 120-kDa protein, which is thought to be the ADAM17/TACE precursor form, Western blotting detected a 100-kDa protein in PDAC cell lines. Maturation of ADAM molecules, including ADAM17/TACE, involves the removal of the prodomain from the precursor to render the metalloproteinase competent (28). The 100-kDa protein has been described as having proteolytic activity. An additional immunoreactive band with a molecular mass of ~20-kDa was detected by the ADAM17/TACE antibody in a few cell lines and correspond to the cleaved cytoplasmic tail of the ADAM molecule because the antibody we used is directed against the cytoplasmic tail. This is supported by a recent observation that the cytoplasmic tail can be cleaved by an autocatalytic process during cell lysis (28).

The subcellular localization, in which the recognition and cleavage of ADAM substrates occurs, is still a matter of debate. Our data show that ADAM17/TACE is expressed on the cell surface of pancreatic cancer cells. Recent data indicate that the cell surface form of ADAM17/TACE is processed and catalytically active (2, 28). In addition, pancreatic cancer cells show a diffuse cytoplasmic immunostaining of endogenous ADAM17/TACE with an accentuation in the perinuclear region of permeabilized pancreatic cancer cells. This is consistent with previous studies in other cell systems (28). Next, we investigated the influence of the TAPI on the proteolytic cleavage of TNF-{alpha} as the main ADAM17/TACE target molecule. Treatment with TAPI dose dependently decreased the amount of soluble TNF-{alpha}. This finding supports the hypothesis that proteolytically active ADAM17/TACE is expressed in PDAC. Although ADAM17/TACE is the most important molecule in the release of TNF-{alpha}, our results cannot exclude the possibility that other molecules might be involved in TNF-{alpha} shedding. Pancreatic cancer cells also express ADAM10 (data not shown),6 which is considered to be an additional candidate for a TNF-{alpha} processing enzyme (30). However, the physiologic TNF-{alpha} shedding activity of ADAM10 is not clear yet. It has been shown that the TNF-{alpha} shedding activity of recombinant ADAM17/TACE was inhibited by the tissue inhibitor of metalloproteinase-3 (TIMP-3; ref. 31) and TIMP-3 expression is down-regulated by hypermethylation of the gene in various types of cancer, including pancreatic cancer (32). Recently, we showed that the expression pattern of ADAM17/TACE and TIMP-3 in prostate cancer samples is inverse of that in specimens of benign prostatic hyperplasia, indicating the potential role of ADAM17/TACE expression in malignancies (33).

Because ADAM17/TACE is widely expressed in PDAC cell lines, we used siRNA-induced gene silencing to investigate its effects on the proliferation and invasion behavior. Our data identify ADAM17/TACE as a key molecule in invasion process of PDAC cells. In contrast to the results in squamous cell carcinoma (17, 18), proliferation of PDAC cells was not significantly influenced by ADAM17/TACE suppression. At present, the molecular mechanism of how ADAM17/TACE increases the invasive potential has not been identified. These effects might be based on the unique potential of ADAM17/TACE to combine adhesion, signaling, and proteolytic activities. Thus, the ADAM17/TACE cleavage of proamphiregulin regulates G protein–coupled, receptor-induced migration in squamous cell carcinoma cells (17, 18). ADAM17/TACE is considered to be a major supplier of EGFR ligands, including TGF-{alpha}, HB-EGF, and amphiregulin in vivo (17, 34). Given the involvement of molecules, such as TGF-{alpha}, TNF receptors, IL-6R, erbB4, and L-selectin in the development of pancreatic cancer (35–37), it seems reasonable to postulate that aberrantly expressed ADAM17/TACE acts as the functional sheddase of these molecules (10–12, 38) in PDAC. ADAM17/TACE is also involved in the expression of MUC1 glycoprotein, which is expressed in most PDACs and characterizes an aggressive phenotype (39). Recently, ADAM17/TACE was shown to function as MUC1 sheddase (40) and thus might contribute to the process of metastasis. The potential biological relevance of the expression and regulation of ADAM17/TACE in cancer progression is supported by the observation that ADAM17/TACE is also involved in EGFR transactivation (17, 18). This seems to be important for tumor cell growth and migration as shown by other authors (17, 18). Considering the recently published data on the interaction of ADAM17/TACE with the {alpha}5ß1 integrin (41), it is also conceivable that ADAM17/TACE may influence the migration and invasion of PDAC cells, independent of metalloproteinase activity. Despite this, the molecular mechanism by which ADAM17/TACE increases the invasive potential in PDAC remains to be defined in further experiments.

Similarly, the regulation of ADAM17/TACE expression and activity is understood only partially. A recent study reported an interaction of ADAM17/TACE with the molecule mitotic arrest deficient 2 (MAD2) and of ADAM9 with a novel MAD2-related protein (MAD2ß; ref. 15). The interaction of ADAM17/TACE and ADAM9 with MAD2 and MAD2ß caused the hypothesis that ADAM molecules might be involved in cell cycle processes. Because a cell cycle–dependent expression of cell cycle–related molecules has been shown (42), we analyzed the ADAM17/TACE mRNA expression in PANC-1 cells arrested in various cell cycle phases. mRNA expression of ADAM17/TACE was cell cycle phase dependent and down-regulated in cells arrested in G2-M phase. Similar to ADAM17/TACE, ADAM9 was also dramatically down-regulated in cells arrested in G2-M phase. Interestingly, ADAM9 has been reported to display aberrant expression in PDAC (43). At present, it is unknown how the ADAMs are involved in cell cycle regulation.

The interaction of pancreatic cancer cells with the surrounding cells and the local concentration of growth factors and/or cytokines may influence the expression of ADAM17/TACE, a mechanism that has been shown recently to act in fibroblasts and PDAC cells (21). These observations, when taken together with our findings about a serum-dependent in vitro up-regulation and a time-dependent down-regulation of ADAM17/TACE mRNA after TNF-{alpha} and IL-6 incubation, indicate possible extracellular regulatory mechanisms. The decreased expression of ADAM17/TACE mRNA after exogenous TNF-{alpha} incubation is in contrast to recent findings in endothelial cells, suggesting a cell system-specific regulation. Furthermore, in contrast to retinal endothelial cells, VEGF did not alter the ADAM17/TACE expression level. The time-dependent regulation by exogenous TNF-{alpha} is of special interest because of the endogenous expression and shedding of TNF-{alpha} in PANC-1 cells. On the other hand, IL-6 concentration is elevated in the serum of PDAC patients and the IL-6R is expressed in pancreatic cancer cells. In this context, it might be important that ADAM17/TACE is known to process the TNF receptors p55 and p75 as well as the IL-6R (10–12, 38).

In summary, we detected the aberrant expression of a proteolytic active ADAM17/TACE in pancreatic cancer cells. The increasing prevalence of ADAM17/TACE expression with higher PanIN grade underlines the role of this molecule in PDAC development. In addition, the gene silencing experiments show a critical role of ADAM17/TACE in the invasion process of PDAC cells. The aberrant expression of proteolytically active ADAM17/TACE may result in an uncontrolled turnover of activated target molecules, such as TNF-{alpha}, TGF-{alpha}, and MUC1 in PDAC. Finally, we showed cell cycle–dependent mRNA expression of ADAM17/TACE.

In conclusion, based on its potential key role in progression of PanINs to PDAC and its malignant behavior, ADAM17/TACE might be an important therapeutic target. Together with our functional data, the blocking of ADAM17/TACE expression and/or the evaluation and development of specific TACE inhibitors might have therapeutic potential even in later stages of cancer. Furthermore, ADAM17/TACE might be useful as a diagnostic marker of pancreatic cancer to distinguish between PDAC and chronic pancreatitis. In summary, aberrant ADAM17/TACE expression might be a diagnostic and therapeutic target in human PDAC.


   Acknowledgments
 
Grant support: NIH grant P50 CA772712 and Marc Lustgarten Foundation for Pancreatic Cancer Research (S.K. Batra).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Erik Moore (University of Nebraska Medical Center) for technical support, Immunotech for giving the TAPI, and K Dege for critical reading of the article.


   Footnotes
 
6 J. Ringel et al., in preparation. Back

Received 9/21/05. Revised 5/12/06. Accepted 6/28/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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