Cancer Research Cancer Health Disparities Conference 2009 Cancer Genome no Abstract
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shintani, Y.
Right arrow Articles by Matsuura, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shintani, Y.
Right arrow Articles by Matsuura, N.
[Cancer Research 64, 4190-4196, June 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Overexpression of ADAM9 in Non-Small Cell Lung Cancer Correlates with Brain Metastasis

Yasushi Shintani1,2, Shigeki Higashiyama3, Mitsunori Ohta2, Hirohisa Hirabayashi2, Sakae Yamamoto1, Tatsuya Yoshimasu4, Hikaru Matsuda2 and Nariaki Matsuura1

1 Department of Molecular Pathology, School of Allied Health Science, Osaka University Faculty of Medicine, Osaka; 2 Department of Surgery, E1, Osaka University Graduate School of Medicine, Osaka; 3 Department of Medical Biochemistry, Ehime University School of Medicine, Ehime; and 4 Department of Thoracic Surgery, Wakayama Medical University, Wakayama, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The "a disintegrin and metalloprotease" (ADAM) family contributes to regulation of the cell–cell and cell–matrix interactions that are critical determinants of malignancy. To determine the relationship between metastasis and ADAM proteins, we compared the mRNA levels of ADAM9, -10, -12, -15, and -17 in sublines of an EBC-1 lung cancer cell line that were highly metastatic to either brain or bone. ADAM9 mRNA levels were significantly higher in highly brain-metastatic sublines than in the parent or highly bone-metastatic sublines. To elucidate the role of ADAM9 in brain metastasis, we stably transfected A549 and EBC-1 cells with a full-length ADAM9 expression vector. Compared with mock-transfectants, ADAM9 overexpression resulted in increased invasive capacity in response to nerve growth factor, increased adhesion to brain tissue, and increased expression of integrin {alpha}3 and ß1 subunits. Administration of the anti-ß1 monoclonal antibody attenuated this increase in invasive and adhesive activity. Intravenous administration of ADAM9-overexpressing A549 cells to mice resulted in micrometastatic foci in the brain and multiple metastatic colonies in the lungs. In contrast, administration of parent and mock-transfected A549 cells to mice resulted in lung tumors without brain metastasis. These results suggest that ADAM9 overexpression enhances cell adhesion and invasion of non-small cell lung cancer cells via modulation of other adhesion molecules and changes in sensitivity to growth factors, thereby promoting metastatic capacity to the brain.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Surgical resection of primary non-small cell lung cancer is frequently followed by tumor recurrence at distant sites as a result of occult micrometastasis (1) . In fact, despite improvements in the diagnosis and treatment of non-small cell lung cancer, the overall survival rate for patients with this disease has remained generally unchanged over the past decade, and most deaths are associated with metastasis to the brain, lungs, and bones (2) .

Metastasis involves a series of events involving tumor cells, including release from the primary tumor, invasion of the surrounding pulmonary parenchyma, and movement into the lymphatic system or vasculature. Those cells that survive within the circulation migrate into parenchymal tissue at distant sites and establish metastatic growth (3) . Invasion into these distant sites requires degradation of extracellular matrix (ECM) components (collagen IV, laminin, and fibronectin), which may be mediated by matrix metalloproteinases (4) . Furthermore, cancer cell adhesion is also an essential cellular function in this process (5) . Integrins are adhesion molecules that are involved in cell–matrix and cell–cell interactions and play a role in the development of tumor metastasis (6 , 7) . Aberrant integrin expression likely contributes to tumor growth and metastasis (8 , 9) .

The recently discovered ADAM family of proteins, so named because they contain a disintegrin and metalloprotease domain, is unique in that the members have the potential to regulate both ECM remodeling and cell migration (10 , 11) . Some ADAM proteins interact with integrins and thus may also play a role in metastasis of cancer cells (12) . In fact, expression of some ADAM proteins is increased in malignant cell populations (13 , 14) , including cells obtained from pancreatic and hepatocellular carcinomas (15 , 16) and from breast cancers (17) . However, the precise role of ADAM proteins in malignancy remains unclear (18) .

Thus, the goal of the present study was to characterize expression of various members of the ADAM family in cancer cell lines with different metastasis profiles, using in vitro and in vivo methods.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Reagents.
A549 and EBC-1 cells were obtained from the Japanese Cancer Research Resources Bank and cultured in tissue culture flasks (Nalge Nunc, Naperville, IL) in DMEM (Nihonseiyaku, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Dainippon Pharmaceutical Co., Tokyo, Japan), 50 units/ml penicillin, and 50 µg/ml streptomycin (Life Technologies, Inc., Frederick, MD). A549 and EBC-1 cells were transfected with ADAM9 cDNA and maintained in DMEM containing 10% FBS and hygromycin (Wako, Osaka, Japan) in concentrations of 600 and 200 µg/ml, respectively.

In vivo selection was carried out to establish a highly metastatic subline of EBC-1, using the procedures described by Fidler (19) . In brief, 1.0 x 106 of the parental EBC-1 cells in 100 µl of serum-free DMEM were injected into the left ventricles of 4-week-old female nude mice. Brain or bone marrow tissues containing the metastatic tumor cells were then excised, minced, and reimplanted into the left ventricle of new recipient mice for the selection of highly metastatic tumor cells. After five rounds of in vivo selection using sequential implantation, metastatic nodules in the tissues were harvested for in vitro culture of metastatic tumor cells.

Quantitation of ADAM mRNA Levels.
Total RNA was extracted, by use of TRIZOL reagent (Life Technologies, Inc., Rockville, IN), from EBC-1 cells plated in 6-cm dishes. Genomic DNA contamination was eliminated by use of DNase (Promega, Madison, WI). Reverse transcription was carried out with 5 µg of total RNA, 0.5 µg of oligo(dT)15, 5 µl of 5x buffer, 2 µl of 2 mM deoxynucleotide triphosphate mixture, 40 units of RNasin, and 5 units of avian myeloblastosis virus reverse transcriptase (Promega). The resulting mixture was incubated at 42°C for 60 min and then at 65°C for 15 min. PCR was carried out in a capillary reaction tube using a reaction mixture containing TaqMan Universal PCR Master Mix (PE Applied Biosystems, Foster City, CA), primer mixture, a labeled probe, and 2 µl of sample cDNA. The concentrations for primer mixtures and probes were 300 nM of each sense and antisense primer and 200 nM of each probe for ADAM mRNA, and 200 nM of each primer and 100 nM of probe for glyceraldehyde-3-phosphate dehydrogenase mRNA (Table 1)Citation . Real-time PCR was run on an ABI PRISM 5700 Sequence Detection System using the following PCR protocol: 50°C for 2 min and 95°C for 10 min, followed by 50 cycles at 95°C for 15 s and 60°C for 1 min. After determination of the threshold cycle, which was defined as the PCR cycle number at which point the fluorescence intensity exceeded the threshold, standard curves for the quantitation of ADAM and glyceraldehyde-3-phosphate dehydrogenase were constructed. This was based on the relationship between the threshold cycle and the results of simultaneous amplification of serial dilutions of the cDNA from parent EBC-1 cells, whose ADAM mRNA level was defined as 1.0.


View this table:
[in this window]
[in a new window]

  		Table 1 Sequence of PCR primers and sequence-specific probes for members of the a-disintegrin-and-metalloprotease (ADAM) family and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

 
The amount of ADAM mRNA in the sample was calculated from the standard curves. To normalize the difference in RNA degradation and RNA loading for RT-PCR in individual samples, we divided the levels of ADAM mRNA by those of glyceraldehyde-3-phosphate dehydrogenase mRNA and defined as relative ADAM expression. Quantitation of ADAM mRNA levels in EBC-1 cells was performed in duplicate and repeated three times.

Western Blot Analysis.
Cells were grown to confluence in 6-cm dishes, washed with PBS, and lysed directly on the dish with lysis buffer containing 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and protease inhibitors on ice for 30 min. The lysates were clarified by centrifugation at 14,000 rpm for 10 min and were mixed with loading sample buffer, heated 5 min at 95°C, separated by SDS-PAGE, and transferred to nitrocellulose. The nitrocellulose filters were blocked with 5% reconstituted dry milk and incubated with the primary antibody overnight at 4°C. Polyclonal antibodies against ADAM9 (Chemicon, Temecula, CA) and ADAM17 (R&D Systems, Minneapolis, MN) were used. The blot membrane was rinsed, incubated with secondary horseradish peroxidase-conjugated antirabbit antibodies, rinsed again, and visualized with an ECL Western blotting detection kit (Amersham Biosciences, Buckinghamshire, United Kingdom).

Generation of A549 and EBC-1 Cell Clones.
Full-length mouse ADAM9 cDNA, the FLAG tag, and the hygromycin resistance gene were subcloned into a pcDNA 3.1 vector. A549 and EBC-1 cells were transfected by use of Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY). For transfection, 1 x 105 cells were plated in 24-well plates for 24 h before transfection in normal growth medium. After the medium was changed to OPTI-MEM I (Invitrogen) without serum, 0.8 µg of DNA and 1.5 µl of Lipofectamine were combined with 100 µl of OPTI-MEM I, incubated at room temperature for 20 min, and added to each well. After incubation at 37°C in a CO2 incubator for 24 h, the cells were collected and plated in 10-cm dishes in DMEM containing 10% FBS and hygromycin. After 3 weeks, stable ring colonies were harvested, and cells expressing ADAM9 were collected by immunoprecipitation analysis. Three clones expressing high levels of ADAM9 were selected and used for experiments. Mock transfection was performed simultaneously with an empty pcDNA 3.1 plasmid.

Cell-Surface Biotinylation and Immunoprecipitation.
Cells were grown to confluence in 6-cm dishes and then washed with PBS and incubated with a non-membrane-permeable biotinylation reagent, sulfo-N-hydroxysuccinimidobiotin (Pierce, Rockford, IL), for 20 min on ice. After quenching with DMEM containing 10% FBS and washing with PBS, the cells were lysed directly on the dish with lysis buffer as described above. The lysates were incubated with anti-FLAG M2 monoclonal antibody (mAb; Sigma, St. Louis, MO) for 4 h at 4°C with rotation. Protein G-Sepharose beads (Amersham Biosciences, Uppsala, Sweden) were added to the extracts and incubated overnight at 4°C with rotation. The beads were then washed three times with lysis buffer, mixed with loading sample buffer, heated for 5 min at 95°C, and separated by SDS-PAGE. The blot membrane was probed with horseradish peroxidase-labeled streptavidin (Vectastain ABC kit; VECTOR Laboratories, Burlingame, CA), and those bound to horseradish peroxidase were visualized.

In Vitro Invasion Assay.
Cell invasion was assessed by Matrigel assay (20) . Chemotaxicell filters (Kurabo, Osaka, Japan) with an 8 µm pore size were precoated with 0.5 mg/ml Matrigel (Becton Dickinson Bioscience, Bedford, MA). Either nerve growth factor (NGF; chemoattractant) or 0.1% BSA (control medium) was added to the bottom chambers of the wells, and cells (2 x 104) were added to the upper chambers. For the blocking assay, cells were preincubated with 10 µg/ml anti-integrin-ß1 mAb (clone P4C10; Chemicon) for 30 min at 37°C, washed, and added to the chambers. After incubation in 10% CO2 for 24 h at 37°C, the filters were removed, fixed with 10% formalin, stained with hematoxylin, and then placed on glass slides after the remaining cells on the upper side were scraped off with a cotton swab. The number of cells that had invaded to the lower side through the pores was counted at x200 magnification in five independent fields.

In Situ Adhesion Assay.
An adhesion assay with brain or lung sections was performed with a Stamper–Woodruff assay (21) . Organs were removed from 4-week-old mice (C57BL/6NCrj) and immediately frozen with liquid N2. Horizontal sections were cut at a thickness of 10 µm. Mock- and ADAM9-transfected cells were labeled with CellTracker green 5-chloromethylfluorescein diacetate (Molecular Probes, Eugene, OR) for 20 min at 37°C. Labeled cells were washed three times with DMEM, resuspended at 3 x 105 cells/ml, mounted on sections, and incubated for 2 h at 37°C with moderate rotation. Unbound cells were washed away with PBS, and the section was fixed, after which the adhered cells were photographed with a confocal laser microscope (LSM-GB200; OLYMPUS, Tokyo, Japan). Cells that had adhered to the frozen tissues were counted at x100 magnification in five independent fields by use of a confocal laser microscope. For the blocking assay, cells were preincubated with 10 µg/ml anti-integrin-ß1 mAb for 30 min at 37°C and then washed and labeled.

Flow Cytometry Analysis.
To stain integrins on the cell surface, cells were harvested, rinsed, suspended in DMEM (supplemented with 1% FBS and 0.03% sodium azide), and sequentially incubated for 30 min at 4°C with mAbs (10 µg/ml) against the human integrin ß1 (Becton Dickinson Bioscience, Bedford, MA), {alpha}1 (Upstate Biotechnology, Lake Placid, NY), {alpha}2 (Chemicon), {alpha}3 (Becton Dickinson Bioscience), and {alpha}6 (Immunotech, Marseille, France) subunits. After washing, the cells were incubated with FITC-conjugated mouse IgG (DAKO, Copenhagen, Denmark) for 30 min at 4°C. Absence of the primary antibody served as a negative control in each case. The cells were washed, resuspended, and analyzed by use of a FACSCalibur (Becton Dickinson Biosciences, Oxnard, CA).

In Vivo Metastasis Assay.
Tumor cells (106 cells/mouse) were injected i.v. into the tail veins of 4-week-old female nude mice (BALB/cABomCr-nu/nu; Charles River Japan, Shiga, Japan), which were cared for in accordance with our institutional guidelines. After 3 months, the mice were euthanized with diethyl ether, and the organs were removed and examined microscopically for metastatic foci after fixation in 10% formalin and staining with H&E or carcinoembryonic antigen (CEA; DAKO, Glostrup, Denmark).

Statistical Analysis.
In vitro invasion assays and in situ adhesion assays were performed in duplicate and repeated three times with three clones of each cell line. Flow cytometry analysis was performed with three clones of each cell line. Mann–Whitney U tests and Kruskal–Wallis tests were performed for comparison of the results (Statview version 5.0 for Windows; Abacus Concepts, Berkeley, CA). P < 0.05 was considered to indicate statistical significance.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ADAM mRNA Levels in EBC-1 Cell Lines.
ADAM mRNA levels in the EBC-1 cell lines are shown in Fig. 1ACitation . ADAM9 mRNA level were significantly higher in the highly brain-metastatic EBC-1 cells than in the parent or the highly bone-metastatic EBC-1 cells.



View larger version (41K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. A, analysis of a disintegrin and metalloprotease (ADAM) mRNA expression in EBC-1 lung cancer cell lines. Absolute ADAM mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels. The Y axis shows ADAM mRNA concentrations relative to those in parent EBC-1 cells. The columns represent mean values of three independent experiments measured in duplicate, and the bars represent the SD. WT, parent EBC-1 cells; EBC-1 Bo, highly bone-metastatic subline of EBC-1; EBC-1 Br, highly brain-metastatic subline of EBC-1. ADAM9 mRNA levels were significantly greater in the highly brain-metastatic subline of EBC-1 compared with wild-type EBC-1 or the highly bone-metastatic subline of EBC-1 (*; P = 0.048, Kruskal–Wallis tests). B, Western blot analysis of ADAM9 and ADAM17 in EBC-1 lung cancer cell lines. The protein sizes (kDa) are indicated to the left of the blot, and the ADAM9 and ADAM17 protein forms are labeled on the right. Although there was no significant difference in ADAM17 expression in EBC-1 cell lines, ADAM9 protein expression was greater in the highly brain-metastatic EBC-1 cell compared with other cell lines.

 
ADAM9 and ADAM17 Protein Expression in EBC-1 Cell Lines.
ADAM9 and ADAM17 protein expression levels in the EBC-1 cell lines are shown in Fig. 1BCitation . Whereas there was no significant difference in ADAM17 expression in EBC-1 cell lines, ADAM9 protein expression was higher in the highly brain-metastatic EBC-1 cells than in the other cell lines.

Inducible Overexpression of ADAM9 in A549 and EBC-1 Cells.
To determine the role of ADAM9 in metastatic potential, overexpression of ADAM9 was induced in A549 and EBC-1 cells (ADAM9-A549 and ADAM9-EBC-1 cells). Cell-surface biotinylation and immunoprecipitation experiments demonstrated the presence of ADAM9 (an 84-kDa fragment in a reduced condition) on the cell surface (Fig. 2)Citation . Three clones overexpressing ADAM9 were selected from each cell line and used for subsequent experiments. Overexpression of ADAM9 had no effect on cell morphology or on proliferation rate (data not shown).



View larger version (31K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. A disintegrin and metalloprotease 9 (ADAM9) detection by immunoprecipitation. After cell-surface biotinylation, immunoprecipitates with anti-FLAG M2 monoclonal antibody showed ADAM9 (84-kDa fragment in a reduced condition) on cell surfaces (arrow). Expression of FLAG-ADAM9 was detected in three clones in each cell line.

 
Overexpression of ADAM9 Alters NGF-Induced Cell Migration and Invasion.
To investigate the role of neurotrophic factors in cell motility, we performed Matrigel assays, using NGF as a chemoattractant. These experiments demonstrated that NGF-induced invasion was significantly greater when ADAM9 transfectants were used compared with mock-transfected cells (Fig. 3)Citation .



View larger version (70K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Nerve growth factor (NGF)-induced invasive activity as assessed by Matrigel assay. A, photograph of A549 cells on the lower side of a Chemotaxicell filter from a Matrigel assay. Chemotaxicell filters were precoated with 0.5 mg/ml Matrigel, and the assay was performed as described in "Materials and Methods." B, invasive activity was determined as the numbers of cells that invaded to the lower side through the filter pores. The columns represent mean values of independent experiments measured in duplicate and repeated three times using three clones of each cell line, and the bars represent the SD. Cell invasion was significantly higher when the ADAM9 transfectants were used compared with the mock-transfected cells. *, P < 0.05 (Mann–Whitney U test). HPF, high-power field.

 
In Situ Adhesion Assay of Frozen Brain Tissues.
To investigate whether ADAM9-overexpressing lung cancer cells exhibited increased adhesion to brain tissues, we performed a cell–brain tissue adhesion assay. Adhesion to brain tissue was significantly higher in ADAM9-A549 and ADAM9-EBC-1 cells than in mock-transfected cells (Fig. 4)Citation .



View larger version (42K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. In situ adhesion assays in brain and lung sections. A, confocal laser microscopy of A549 cells labeled with CellTracker green 5-chloromethylfluorescein diacetate in brain or lung tissues. Mock- and ADAM9-transfected cells are stained green, and the nuclei of brain or lung cells are stained red by propidium iodide. Bar = 100 µm. B, adhesive activity was determined as the numbers of cells that adhered to the frozen tissues as described in "Materials and Methods." The columns represent mean values of independent experiments measured in duplicate and repeated three times using three clones of each cell line, and the bars represent the SD. Adhesion to brain sections was significantly increased in a disintegrin and metalloprotease 9 (ADAM9)-overexpressing cells compared with mock-transfected cells (*, P < 0.05, Mann–Whitney U test), whereas there was no difference in adhesion to the lung tissue sections between the cell lines. HPF, high-power field.

 
Integrin Expression in Transfectant Cells.
To further characterize the relationship between ADAM9 and invasive and adhesive capacity in brain tissues, we assessed expression of integrins. Cell-surface expression of integrin {alpha}3 and ß1 subunits was higher in ADAM9-A549 and ADAM9-EBC-1 cells than in mock-transfected cells (Fig. 5)Citation . However, expression of the {alpha}1, {alpha}2, and {alpha}6 subunits was similar among all cell lines. Preincubation of cells with the integrin-ß1-blocking mAb resulted in an attenuation of invasive activity (Fig. 6A)Citation and adhesion to brain tissues (Fig. 6, B and C)Citation .



View larger version (45K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. A, flow cytometric analysis of expression of human integrin ß1, {alpha}1, {alpha}2, {alpha}3, and {alpha}6 in A549 and EBC-1 cells. The thin lines represent the integrin expression of mock transfectants, and the thick lines represent the integrin expression of a disintegrin and metalloprotease 9 (ADAM9) transfectants. B, expression of human integrin ß1 and {alpha}3 in A549 and EBC-1 cells. The Y axis shows mean fluorescence intensity for each integrin. The columns represent mean values of independent experiments using three clones of each cell line, and the bars represent the SD. Expression of integrin {alpha}3 and ß1 was significantly greater in ADAM9 transfectants than in mock transfectants (*, P < 0.05, Mann–Whitney U test).

 


View larger version (56K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. A, suppression of invasive activity by inhibition of integrin ß1. Invasive activity was determined as the numbers of cells that invaded to the lower side through the filter pores as described in "Materials and Methods." The columns represent mean values of independent experiments measured in duplicate and repeated three times using three clones of each cell line, and the bars represent the SD [(+), with anti-integrin-ß1 monoclonal antibody (mAb); (–), without anti-integrin-ß1 mAb]. The nerve growth factor-induced invasive activity of a disintegrin and metalloprotease 9 (ADAM9)-overexpressing cells was reduced to control levels after treatment with the integrin-ß1-blocking mAb (*, P < 0.05, Mann–Whitney U test). B, suppression of in situ adhesion assay by inhibition of integrin ß1. Confocal laser microscopy of EBC-1 cells labeled with CellTracker green 5-chloromethylfluorescein diacetate on the brain tissues. Mock- and ADAM9-transfected cells are stained green, and the nuclei of brain cells are stained red by propidium iodide, as described in "Materials and Methods." C, adhesive activity was determined as the number of cells that adhered to the frozen tissues as described in "Materials and Methods." The columns represent mean values of independent experiments measured in duplicate and repeated three times using three clones of each cell line, and the bars represent the SD. Preincubation of the cells with the integrin-ß1-blocking mAb led to a significant decrease in adhesion activity (*, P < 0.05, Mann–Whitney U test). HPF, high-power field.

 
Overexpression of ADAM9 Induces Brain Metastasis: In Vivo Metastasis Assay.
Twelve weeks after i.v. administration of parent A549, mock-transfected A549, and ADAM9-transfected A549 cells, multiple tumor foci were seen in the lung tissues of all animals (Table 2Citation ; Fig. 7, A and BCitation ). There were no differences in the number of metastatic colonies in the lungs of animals injected with parent A549 cells, mock-transfectants, or ADAM9-transfectants. Although no tumors were observed in other organs in animals that received parent A549 cells (n = 5) or mock-A549 cells (n = 5), H&E staining and CEA immunostaining demonstrated the presence of microscopic metastasis in the brains of 15 of 18 mice receiving injections of ADAM9-A549 cells (Fig. 7, C–F)Citation . Because CEA is not expressed in mouse cells, positive CEA immunostaining in these cases suggests that the microscopic foci are composed of human cancer cells. Some mice receiving injections of ADAM9-A549 cells demonstrated neurological symptoms (e.g., paralysis), which is consistent with the presence of brain metastasis.


View this table:
[in this window]
[in a new window]

  		Table 2 Metastasis of A549 wild-type, mock-transfected A549, and a-disintegrinand-metalloprotease-9 (ADAM9)-transfected A549 cells after i.v. administration to nude mouse

 


View larger version (104K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 7. In vivo metastasis assay. Five or six animals in each group of nude mice received an i.v. injection of 106 cells. After 3 months, the mice were euthanized, and their organs were removed and examined. A, mice receiving A549 cells developed multiple colonies in the lungs (arrows). B, microscopic appearance of metastatic foci in the lungs of the mouse receiving A549 cells. (H&E staining; x40 magnification). C and D, microscopic appearance of micrometastatic foci in the brain of the mice receiving a disintegrin and metalloprotease 9 (ADAM9)-A549 cells. Mice receiving ADAM9-overexpressing A549 cells developed multiple micrometastatic foci (arrows in C) in the cerebral cortex (H&E staining; magnification, C, x40; D, x100). E, immunoperoxidase staining shows carcinoembryonic antigen localization in the metastatic lesion exclusively (magnification, x100). F, micrometastatic cancer cells near the brain blood vessel (H&E staining; magnification, x100).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
ADAM9 is a widely expressed, catalytically active metalloprotease–disintegrin protein that is highly conserved in humans and mice (22) . The present study demonstrates that ADAM9 mRNA levels are significantly higher in the highly brain-metastatic EBC-1 subline and that ADAM9 overexpression in A549 and EBC-1 cells resulted in an increase in NGF-induced invasive capacity and cell adhesion to brain tissue. Furthermore, i.v. injection of ADAM9-overexpressing A549 cells into nude mice resulted in brain metastasis. Together, these data suggest that increased expression of ADAM9 correlates with invasive and adhesive potential and that ADAM9 participates in the induction of tumor cell trafficking to the brain. This is the first study to demonstrate a relationship between cancer metastasis and ADAM9 expression.

Members of the ADAM family, including ADAM9, -10, -12, -15, and -17, contain the amino acid sequence HEXXHXXGXXH in the catalytic zinc-binding site, which confers the functional metalloprotease capacity. Specifically, the recombinant metalloprotease domain of ADAM9 shows activity against gelatin, ß-casein, and fibronectin (23) . Therefore, it is likely that this metalloprotease domain plays a role in the ECM degradation or basement membrane modifications required for successful metastasis (13) . Indeed, in the present study, the increased invasive capability of ADAM9-A549 and ADAM9-EBC-1 cells was demonstrated by Matrigel assay. ADAM9 may either directly degrade the ECM or induce activation of other ECM proteases, such as matrix metalloproteinases, thereby allowing tumor cell penetration into the matrix of the brain.

Increased expression of integrin ß1 also results in increased cell invasion and metastasis in various tumor types, including malignant melanoma, malignant glioma, ovarian cancer, colorectal cancer, and breast cancer (24, 25, 26, 27) . Integrin {alpha}3ß1 is associated with gelatinase B (matrix metalloproteinase-9) activity (28) . In the present study, integrin {alpha}3ß1 expression was up-regulated in ADAM9-overexpressing cells, and preincubation of cells with the integrin-ß1-blocking mAb resulted in a decrease in invasive activity. These results suggest that up-regulation of integrin {alpha}3ß1 may play an important role in the invasive potential of ADAM9-overexpressing cells.

A plasma membrane-anchored disintegrin domain may play a role in cell–cell interactions by functioning as an integrin ligand (29 , 30) . Recent studies reported that ADAM9 and integrin ß1 colocalize (31) and that ADAM9 regulates the motility of cells by binding to integrin {alpha}6ß1 (32 , 33) . These integrin-regulating functions make ADAM9 an ideal candidate to serve as an adhesion- and migration-regulating molecule (34) . Taken together, these data suggest that ADAM9 regulates tumor adhesion and invasion to brain tissues through modulation of integrin function in cancer cells.

Metastasis to the brain requires that malignant cells undergo a series of events, including attachment to the endothelial cells of the brain microvessels, activation by brain endothelial cell-derived motility factors and brain-derived invasion factors, invasion through the blood–brain barrier, and activation by brain-derived survival and growth factors (35) . Paget (36) suggested that certain tumor cells (seeds) have a specific affinity for the milieu of certain organs (soil) and that metastasis occurs only when the seeds and soil are compatible. This implies that metastasis depends on interactions between tumor cells and the organ microenvironment. In the present study, ADAM9-A549 cells survived, invaded, and proliferated within the brain, indicating that they are compatible with the local brain microenvironment.

The proliferation of human lung cancer is regulated by several growth factors, both in vivo and in vitro, via an autocrine or paracrine mechanism (37) . NGF enhances cancer cell growth and invasion in malignant melanoma, pancreatic cancer, prostate cancer, and lung cancer cells (38, 39, 40, 41) , whereas TrkA, a NGF high-affinity receptor recognized by a tyrosine-specific protein kinase, may modulate the biological activity of NGF in lung neoplasm (37) . In the present study, ADAM9 transfectants showed an increase in NGF-induced invasion compared with mock-transfected cells. These data suggest that NGF functions as a chemoattractant to ADAM9-transfected cells and promotes tumor cell penetration into the matrix of the neurotrophin-rich stromal brain microenvironment.

In conclusion, ADAM9 overexpression enhanced cancer cell adhesion and invasion via modulation of other adhesion molecules and changes in sensitivity to growth factors. Furthermore, overexpression of ADAM9 promoted cancer cell trafficking to the brain.


   ACKNOWLEDGMENTS
 
We thank Dr. Hiroshi Ishiguro at the Institute for Comprehensive Medical Science, Fujita Health University (Aichi, Japan) for providing the mouse ADAM9 expression plasmid.


   FOOTNOTES
 
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.

Requests for reprints: Nariaki Matsuura, Department of Molecular Pathology, School of Allied Health Science, Osaka University Faculty of Medicine, 1-7 Yamadaoka, Suita, Osaka 565-0817, Japan. Phone: 81-6-6879-2595; Fax: 81-6-6879-2499; E-mail: matsuura{at}sahs.med.osaka-u.ac.jp

Received 10/15/03. Revised 2/29/04. Accepted 3/17/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Fry WA, Menck HR, Winchester DP The national cancer data base report on lung cancer. Cancer (Phila), 77: 1947-55, 1996.
  2. Yano T, Yokoyama H, Inoue T, et al The first site of recurrence after complete resection in non-small-cell carcinoma of the lung. Comparison between pN0 disease and pN2 disease. J Thorac Cardiovasc Surg, 108: 680-3, 1994.[Abstract/Free Full Text]
  3. Albelda SM Role of integrins and other cell adhesion molecules in tumor progression and metastasis. Lab Investig, 68: 4-17, 1993.[Medline]
  4. Murphy G, Docherty AJ The matrix metalloproteinases and their inhibitors. Am J Respir Cell Mol Biol, 7: 120-5, 1992.
  5. Kohn EC, Liotta LA Molecular insights into cancer invasion: strategies for prevention and intervention. Cancer Res, 55: 1856-62, 1995.[Abstract/Free Full Text]
  6. Hynes RO Integrins: versatility, modulation, and signaling in cell adhesion. Cell, 69: 11-25, 1992.[CrossRef][Medline]
  7. Juliano RL, Varner JA Adhesion molecules in cancer: the role of integrins. Curr Opin Cell Biol, 5: 812-8, 1993.[CrossRef][Medline]
  8. Smythe WR, Wasfi D, Bavaria JE, Albelda SM, Kaiser LR Loss of alpha-v integrin expression and recurrence in node-negative lung carcinoma. Ann Thorac Surg, 64: 949-53, 1997.[Abstract/Free Full Text]
  9. Adachi M, Taki T, Huang CL, et al Reduced integrin alpha3 expression as a factor of poor prognosis of patients with adenocarcinoma of the lung. J Clin Oncol, 16: 1060-7, 1998.[Abstract]
  10. Wolfsberg TG, Primakoff P, Myles DG, White JM ADAM, a novel family of membrane proteins containing A Disintegrin And Metalloprotease domain: multipotential functions in cell-cell and cell-matrix interactions. J Cell Biol, 131: 27578 1995.
  11. Black RA, White JM ADAMs: focus on the protease domain. Curr Opin Cell Biol, 10: 654-9, 1998.[CrossRef][Medline]
  12. Almeida EA, Huovila AP, Sutherland AE, et al Mouse egg integrin alpha 6 beta 1 functions as a sperm receptor. Cell, 81: 1095-104, 1995.[CrossRef][Medline]
  13. Wu E, Croucher PI, McKie N Expression of members of the novel membrane linked metalloproteinase family ADAM in cells derived from a range of haematological malignancies. Biochem Biophys Res Commun, 235: 437-42, 1997.[CrossRef][Medline]
  14. Iba K, Albrechtsen R, Gilpin BJ, Loechel F, Wewer UM Cysteine-rich domain of human ADAM 12 (meltrin alpha) supports tumor cell adhesion. Am J Pathol, 154: 1489-501, 1999.[Abstract/Free Full Text]
  15. Grutzmann R, Foerder M, Alldinger I, et al Gene expression profiles of microdissected pancreatic ductal adenocarcinoma. Virchows Arch, 443: 508-17, 2003.[CrossRef][Medline]
  16. Tannapfel A, Anhalt K, Hausermann P, et al Identification of novel proteins associated with hepatocellular carcinomas using protein microarrays. J Pathol, 201: 238-49, 2003.[CrossRef][Medline]
  17. O’Shea C, McKie N, Buggy Y, et al Expression of ADAM-9 mRNA and protein in human breast cancer. Int J Cancer, 105: 754-61, 2003.[CrossRef][Medline]
  18. McCulloch DR, Harvey M, Herington AC The expression of the ADAMs proteases in prostate cancer cell lines and their regulation by dihydrotestosterone. Mol Cell Endocrinol, 167: 11-21, 2000.[CrossRef][Medline]
  19. Fidler IJ Selection of successive tumour lines for metastasis. Nat New Biol, 242: 148-9, 1973.[Medline]
  20. Albini A, Iwamoto Y, Kleinman HK, et al A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res, 47: 3239-45, 1987.[Abstract/Free Full Text]
  21. Stamper HB, Jr, Woodruff JJ Lymphocyte homing into lymph nodes: in vitro demonstration of the selective affinity of recirculating lymphocytes for high-endothelial venules. J Exp Med, 144: 828-33, 1976.[Abstract/Free Full Text]
  22. Weskamp G, Cai H, Brodie TA, et al Mice lacking the metalloprotease-disintegrin MDC9 (ADAM9) have no evident major abnormalities during development or adult life. Mol Cell Biol, 22: 1537-44, 2002.[Abstract/Free Full Text]
  23. Schwettmann L, Tschesche H Cloning and expression in Pichia pastoris of metalloprotease domain of ADAM 9 catalytically active against fibronectin. Protein Expr Purif, 21: 65-70, 2001.[CrossRef][Medline]
  24. Fujita S, Watanabe M, Kubota T, Teramoto T, Kitajima M Alteration of expression in integrin beta 1-subunit correlates with invasion and metastasis in colorectal cancer. Cancer Lett, 91: 145-9, 1995.[CrossRef][Medline]
  25. Paulus W, Baur I, Beutler AS, Reeves SA Diffuse brain invasion of glioma cells requires beta 1 integrins. Lab Invest, 75: 819-826, 1996.[Medline]
  26. Fukushima Y, Ohnishi T, Arita N, Hayakawa T, Sekiguchi K Integrin alpha3beta1-mediated interaction with laminin-5 stimulates adhesion, migration and invasion of malignant glioma cells. Int J Cancer, 76: 63-72, 1998.[CrossRef][Medline]
  27. Arboleda MJ, Lyons JF, Kabbinavar FF, Bray MR, Snow BE, Ayala R, Danino M, Karlan BY, Slamon DJ Overexpression of AKT2/protein kinase Bbeta leads to up-regulation of beta1 integrins, increased invasion, and metastasis of human breast and ovarian cancer cells. Cancer Res, 63: 196-206, 2003.[Abstract/Free Full Text]
  28. Morini M, Mottolese M, Ferrari N, et al The alpha 3 beta 1 integrin is associated with mammary carcinoma cell metastasis, invasion, and gelatinase B (MMP-9) activity. Int J Cancer, 87: 336-42, 2000.[CrossRef][Medline]
  29. Weskamp G, Kratzschmar J, Reid MS, Blobel CP MDC9, a widely expressed cellular disintegrin containing cytoplasmic SH3 ligand domains. J Cell Biol, 132: 717-26, 1996.[Abstract/Free Full Text]
  30. Yagami-Hiromasa T, Sato T, Kurisaki T, Kamijo K, Nabeshima Y, Fujisawa-Sehara A A metalloprotease-disintegrin participating in myoblast fusion. Nature (Lond), 377: 652-6, 1995.[CrossRef][Medline]
  31. Mahimkar RM, Baricos WH, Visaya O, Pollock AS, Lovett DH Identification, cellular distribution and potential function of the metalloprotease-disintegrin MDC9 in the kidney. J Am Soc Nephrol, 11: 595-603, 2000.[Abstract/Free Full Text]
  32. Nath D, Slocombe PM, Webster A, Stephens PE, Docherty AJ, Murphy G 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, 113: 2319-28, 2000.[Abstract]
  33. Chen MS, Almeida EA, Huovila AP, et al Evidence that distinct states of the integrin alpha6beta1 interact with laminin and an ADAM. J Cell Biol, 144: 549-61, 1999.[Abstract/Free Full Text]
  34. Al-Fakhri N, Wilhelm J, Hahn M, et al Increased expression of disintegrin-metalloproteinases ADAM-15 and ADAM-9 following upregulation of integrins alpha5beta1 and alphavbeta3 in atherosclerosis. J Cell Biochem, 89: 808-23, 2003.[CrossRef][Medline]
  35. Nicolson GL, Menter DG Trophic factors and central nervous system metastasis. Cancer Metastasis Rev, 14: 303-21, 1995.[CrossRef][Medline]
  36. Paget S The distribution of secondary growths in cancer of the breast. Lancet, 1: 571-3, 1889.
  37. Ricci A, Greco S, Mariotta S, et al Neurotrophins and neurotrophin receptors in human lung cancer. Am J Respir Cell Mol Biol, 25: 439-46, 2001.[Abstract/Free Full Text]
  38. Marchetti D, Menter D, Jin L, Nakajima M, Nicolson GL Nerve growth factor effects on human and mouse melanoma cell invasion and heparanase production. Int J Cancer, 55: 692-9, 1993.[Medline]
  39. Zhu Z, Kleeff J, Kayed H, et al Nerve growth factor and enhancement of proliferation, invasion, and tumorigenicity of pancreatic cancer cells. Mol Carcinog, 35: 138-47, 2002.[CrossRef][Medline]
  40. Krygier S, Djakiew D Neurotrophin receptor p75(NTR) suppresses growth and nerve growth factor-mediated metastasis of human prostate cancer cells. Int J Cancer, 98: 1-7, 2002.[CrossRef][Medline]
  41. Oelmann E, Sreter L, Schuller I, et al Nerve growth factor stimulates clonal growth of human lung cancer cell lines and a human glioblastoma cell line expressing high-affinity nerve growth factor binding sites involving tyrosine kinase signaling. Cancer Res, 55: 2212-9, 1995.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 JNCI J Natl Cancer InstHome page
J. Yuan, J. Ma, H. Zheng, T. Shi, W. Sun, Q. Zhang, D. Lin, K. Zhang, J. He, Y. Mao, et al.
Overexpression of OLC1, Cigarette Smoke, and Human Lung Tumorigenesis
J Natl Cancer Inst, November 19, 2008; 100(22): 1592 - 1605.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. J. Najy, K. C. Day, and M. L. Day
The Ectodomain Shedding of E-cadherin by ADAM15 Supports ErbB Receptor Activation
J. Biol. Chem., June 27, 2008; 283(26): 18393 - 18401.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
E. R. Gerstner and R. L. Fine
Increased Permeability of the Blood-Brain Barrier to Chemotherapy in Metastatic Brain Tumors: Establishing a Treatment Paradigm
J. Clin. Oncol., June 1, 2007; 25(16): 2306 - 2312.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S.-Y. Sung, H. Kubo, K. Shigemura, R. S. Arnold, S. Logani, R. Wang, H. Konaka, M. Nakagawa, S. Mousses, M. Amin, et al.
Oxidative Stress Induces ADAM9 Protein Expression in Human Prostate Cancer Cells
Cancer Res., October 1, 2006; 66(19): 9519 - 9526.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
A. Mazzocca, R. Coppari, R. De Franco, J.-Y. Cho, T. A. Libermann, M. Pinzani, and A. Toker
A Secreted Form of ADAM9 Promotes Carcinoma Invasion through Tumor-Stromal Interactions
Cancer Res., June 1, 2005; 65(11): 4728 - 4738.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shintani, Y.
Right arrow Articles by Matsuura, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shintani, Y.
Right arrow Articles by Matsuura, N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online