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Cell and Tumor Biology |
1 Institute of Molecular Pathology, University of Copenhagen; 2 Department of Pathology, Rigshospitalet University Hospital, Copenhagen, Denmark; 3 Department of Pathology, Turku University, Turku, Finland; and 4 Beth Israel Deaconess Center, Harvard Medical School, Boston, Massachusetts
Requests for reprints: Ulla M. Wewer, Institute of Molecular Pathology, University of Copenhagen, Frederik V's vej 11, 2100 Copenhagen, Denmark. Phone: 45-3532-6056; Fax: 45-3532-6081; E-mail: ullaw{at}pai.ku.dk.
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Introduction |
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30% died within the first postnatal week for unknown reasons (5). Mice surviving beyond the first postnatal week seemed normal, but some had deficiencies in development of interscapular brown adipose tissue as well as reduced neck and interscapular muscle tissue (5). In a subsequent study, we showed that mice overexpressing ADAM12 exhibit increased adipogenesis (6). In humans, two alternatively spliced forms of ADAM12 are expressed, the prototype transmembrane form, ADAM12-L, and a shorter secreted form, ADAM12-S (7). Interestingly, both forms are highly expressed in placenta (7) and levels of ADAM12 in the serum of pregnant women seem to be a valuable marker for abnormal placental growth and could potentially serve as a useful marker in clinical prenatal diagnostics (8). ADAM12 is expressed at low levels in most normal adult tissues, but it is expressed at higher levels in a large proportion of human carcinomas, including breast, gastric, colon carcinomas, and liver metastases (9–11). A recent proteomic approach identified ADAM12 in the urine of breast cancer patients and showed that its levels in urine correlated with tumor progression, suggesting that ADAM12 could be used as a noninvasive diagnostic test for breast cancer (12). ADAM12 expression in human glioblastomas was shown to correlate with cell proliferation and shedding of pro-heparin-binding epidermal growth factor (pro-HB-EGF; ref. 13). Finally, it should be mentioned that microarray analysis showed that ADAM12 is up-regulated in aggressive fibromatosis (14).
In the present study, we investigated the function of ADAM12 in breast cancer. We show that ADAM12 promotes tumor progression by a mechanism involving the ability of ADAM12 to differentially influence the apoptotic potential of tumor versus stromal cells.
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Materials and Methods |
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The experimental use of the surgical specimens was in accordance with the guidelines of the Danish Breast Cancer Group and the hospital ethical guidelines of Turku University Hospital and Jyväskylä Central Hospital, respectively.
Transgenic mice. To generate mammary gland–specific ADAM12 transgene expression, a modified version of the mouse mammary tumor virus long terminal repeat promoter/enhancer (MMTV-LTR)-Sv40-Bssk vector (a generous gift from R.G. Oshima, The Burnham Institute, La Jolla, CA) was used. Briefly, the EcoRI site of the vector's multiple cloning site was replaced by PacI. cDNAs encoding either the transmembrane human ADAM12-L lacking the cytoplasmic tail (ADAM12-
cyt) or the secreted form of human ADAM12 (ADAM12-S) were cloned into the PacI site of Sv40-Bssk vector. PvuI and SpeI were used to excise MMTV-ADAM12-S and MMTV-ADAM12-cyt fragments, which were injected into the male pronucleus of fertilized zygotes isolated from superovulated donor mice. Injected embryos were implanted into pseudopregnant recipients and allowed to develop to term. For the ADAM12-S transgenic mice, C57BL/6J x CBA, F1 mice were used as donors and recipients and for backcrossing, whereas the inbred mouse strain, FVB/n, was used for the MMTV-ADAM12-cyt mice. C57BL/6J and FVB/n mice were purchased from M&B (Copenhagen, Denmark).
Heterozygous transgenic FVB/N-TgN(MMTV-PyMT)634Mul mice expressing polyomavirus middle T antigen (PyMT) in the mammary gland under transcriptional control of the MMTV-LTR were obtained from The Jackson Laboratory (Bar Harbor, ME). MMTV-PyMT males were crossed with females from the transgenic lines MMTV-ADAM12-S (C57BL/6J x CBA, F1) and MMTV-ADAM12-cyt (FVB/n) to generate ADAM12-S/PyMT and ADAM12-cyt/PyMT mice, respectively, and their corresponding PyMT littermates. All animals analyzed in this study were females.
The MMTV-ADAM12-S and MMTV-ADAM12-cyt transgenic mice were identified by PCR on tail genomic DNA using the human ADAM12 forward primer 5'-CAGGATCCAGAGAGACCCTCAAG-3' and the reverse primer 5'-CAACTCGAGCGGCAGGTTAAACAG-3'. The PyMT transgene was identified using the sense primer 5'-GGAAGCAAGTACTTCACAAGGG-3' and the antisense primer 5'-GGAAAGTCACTAGGAGCAGGG-3'.
After weaning, all animals were both weighed and examined for mammary gland tumors by palpation twice a week. The date that the first tumor appeared was noted. Mice were sacrificed at 12 weeks of age. One hour before sacrifice, 5-bromo-2'-deoxyuridine (BrdUrd; 50 µg/g body weight) was injected i.p. The tumors were dissected, weighed, and divided into pieces, which were either frozen in liquid nitrogen or fixed in formalin for further analysis. The lungs were removed, fixed in formalin, and embedded in paraffin for further evaluation of metastasis.
All experiments were conducted in accordance with the guidelines of the Animal Experiment Inspectorate, Denmark.
Cell culture. Mouse 3T3-L1 fibroblastic preadipocytes (CL-173), CHO-K1 Chinese hamster ovarian cells (CCL-61), human MG-63 osteosarcoma (CRL-1427), human A-431 squamous cell carcinoma (CRL-1555), human HeLa adenocarcinoma (CCL-2), and normal human lung fibroblasts MRC-5 (CCL-171) were obtained from the American Type Culture Collection (Rockville, MD). HEK293-EBNA cells were obtained from Invitrogen (Tåstrup, Denmark). All cell lines except CHO-K1 were cultured in DMEM with Glutamax I and 4,500 mg/L glucose, 50 units/mL penicillin, 50 µg/mL streptomycin, and 10% fetal bovine serum (FBS); for MRC-5 cells, 1 mmol/L sodium pyrovate (Invitrogen) was also included in the culture media. CHO-K1 cells were maintained in DMEM/F12 (1:1; Invitrogen) supplemented with 50 units/mL penicillin, 50 µg/mL streptomycin, and 10% FBS. 3T3-L1 cells stably expressing hADAM12-cyt or vector control were generated by retroviral transduction as described previously (15) and kept in selection media (2.0 µg/mL puromycin). CHO-K1 cells were transfected using the FuGene 6 Transfection Reagent (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions.
Antibodies and reagents. Monoclonal antibodies (6E6, 8F8, 6C10) and polyclonal antiserum (rb 122, rb 109) to ADAM12 were generated as previously described (7, 16). Rabbit monoclonal antibodies to cyclin D1 (clone SP4) were obtained from Lab Vision (Fremont, CA); rabbit polyclonal IgG to Bax (sc-493) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); monoclonal antibody to actin (MAB 1501R) was purchased from Chemicon (Temecula, CA); rabbit anti-BrdUrd antibody was obtained from Roche (Indianapolis, IN); and rabbit antibody to active caspase3 (ab2302) was purchased from Abcam, Ltd. (Cambridge, United Kingdom). Secondary antibodies were obtained from Dako A/S (Glostrup, Denmark). Recombinant ADAM12-S was generated by transfecting human HEK293-EBNA cells with cDNA encoding human ADAM12-S and purified using fast protein liquid chromatography cation exchange and concanavalin A affinity chromatography as described previously (17). Recombinant human tumor necrosis factor (TNF) 
and mouse mTNF were purchased from Strathmann Biotec AG (Hamburg, Germany). Cycloheximide and BrdUrd were purchased from Sigma-Aldrich (Brøndby, Denmark). The broad caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-CH2F (zVAD-fmk) was obtained from Bachem (Bubendorf, Switzerland).
Histologic analysis and immunohistochemical staining. Tissue samples were fixed in formalin and embedded in paraffin; 4 µm sections were stained with H&E for morphologic analysis. Immunostaining procedures were done as previously described (10). To examine tissue sections for apoptosis, ApoTag reagents were used according to the manufacturer's instructions (Invitrogen). Each tissue section was scanned, enlarged, and the approximate area of the tissue section estimated using square transparent paper overlay imprinted with squares measuring 25 mm2. The numbers of apoptotic and necrotic cells present in each section of tissue were counted and the numbers per mm3 estimated essentially as previously described (18).
Samples from mammary gland tumors were obtained from 10 ADAM12-S/PyMT compared with 10 PyMT littermates and 10 ADAM12-cyt/PyMT compared with 10 PyMT littermates and processed as described above. Malignant (early and late carcinoma) and premalignant (hyperplasia and adenoma) areas were marked and the areas estimated. The measurements in both cases were carried out independently by two observers.
Western blotting. Breast tumor tissue from humans and mice and cell extracts were examined by Western blotting as described previously (6, 15, 16). In brief, proteins were extracted in radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 7.4), 1% Triton X-100, 25 mmol/L HEPES, 150 mmol/L NaCl, 0.2% deoxycholate, 5 mmol/L MgCl2, 1 mmol/L Na3VO4, 1 mmol/L NaF, and a protease inhibitor cocktail (complete, EDTA-free protease inhibitor cocktail tablets; Roche Molecular Biochemical, Hvidovre, Denmark)]. In some cases, immunoprecipitation with monoclonal antibodies (6E6, 8F8, 6C10) and protein-G sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden) was done. Detection of ADAM12 was done using rb 122 as a primary antibody, horseradish peroxidase–conjugated secondary antibody, and the chemiluminescence SuperSignal reagent (Pierce, Cheshire, United Kingdom). Detection of Bax and actin was done by Western blotting using specific primary antibodies as described above.
Construction of plasmids for in vitro experiments. cDNAs encoding human ADAM12-L truncated at nucleotide 2,498, resulting in a membrane-inserted protein lacking the cytoplasmic tail (ADAM12-cyt), and ADAM12-cyt with a E351-Q catalytic site mutation were cloned into the pEGFP-N1 vector (Clontech Laboratories, Heidelberg, Germany) to create COOH-terminal fusion proteins with enhanced green fluorescence protein (EGFP) or into pBABEpuro for retroviral transduction as previously described (15). cDNA for ADAM12-S was cloned into the pCEP4 vector (Invitrogen) for episomal expression in HEK293-EBNA cells.
Cytotoxicity assay and detection of condensed nuclei of cultured cells. Apoptosis was induced by treating cells with varying concentrations of TNF in the presence of 5 µmol/L cycloheximide for 24 hours, except for 3T3-L1 cells that were treated for 5 hours. Alternatively, cell media was removed and the cells were subjected to UVC irradiation (254 nm) with 60 J/m2. After irradiation, fresh media was added and the cells were incubated for 24 hours. Cell cytotoxicity was analyzed using the Cytotoxicity Detection kit (Roche) as described by the manufacturer. Briefly, 5,000 to 10,000 cells were cultured on 96-well plates 24 hours before induction. Following induction, cytotoxicity was quantitated by measuring the lactate dehydrogenase activities in the culture media (M) and in the attached cells after lysis in media with 1% Triton X-100 (A). Calculation of cytotoxicity was done using the ratio M/(M + A). Nuclear condensation was detected by staining the cells with membrane permeable Hoechst 33342 (Molecular Probes, Leiden, the Netherlands). To avoid washing away poorly attached cells, media was carefully removed from the cell layer. Next, cells were fixed and stained simultaneously by adding 4% paraformaldehyde in PBS containing 2.5 µg/mL Hoechst 33342 and incubating for 30 minutes at room temperature. Finally, cells were washed once with cold PBS before visualization by fluorescence microscopy and counting the percentage of cells with condensed and brightly stained nuclei. When counting the percentage of green cells with condensed nuclei, >100 cells were counted. When counting the percentage cells with condensed nuclei out of all cells, at least four different fields were counted. The numbers represent independent results from two individuals that were obtained in a blinded manner in at least two independent experiments.
Annexin V/propidium iodide analysis. HeLa cells were treated with purified recombinant ADAM12-S or vehicle for 24 hours followed by 18-hour treatment with 10 ng/mL hTNF and 5 µmol/L cycloheximide in the presence of recombinant ADAM12-S. The percentage of apoptotic cells was determined using an annexin V-FITC detection kit (Oncogene, Inc., Boston, MA) according to manufacturer's instructions. Briefly, cells were trypsinized, resuspended in the culture medium, and washed in PBS. Cells were stained with annexin V-FITC for 15 minutes and washed; propidium iodide was then added before the cells were analyzed with a FACScalibur machine (BD Biosciences, Palo Alto, CA). The cells were kept on ice until the fluorescence-activated cell sorting (FACS) analysis was completed.
Cell proliferation assays in vitro. DNA synthesis was determined using the 5-bromo-2'-deoxyuridine labeling and detection kit from Boehringer Mannheim (Mannheim, Germany). Briefly, cells were incubated for 60 minutes at 37°C with 10 µmol/L BrdUrd and subsequently fixed in methanol. DNA was denatured by incubating in 2 N HCl for 15 minutes followed by neutralization in 0.1 mol/L borate buffer (pH 8.5). BrdUrd incorporation was detected using an anti-BrdUrd antibody as described by the manufacturer. Short-term growth rates were determined by seeding 10,000 cells/well in 24-well plates. At days 1, 2, 3, and 4 after seeding, cells in four individual wells were trypsinized and counted. Trypan blue dye (0.4% w/v) exclusion was used to ensure equal viability of the cells.
Statistical analysis. Statistical analysis was done using Kaplan-Meier to determine tumor-free periods, and the log-rank test, the Mann Whitney test, or the Student's t test for comparing two independent groups. P < 0.05 were considered statistically significant.
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Results and Discussion |
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cyt; ref. 19) and the other expressing the secreted form, ADAM12-S, mimicking the finding of ADAM12-S in the urine of breast cancer patients (12). MMTV-ADAM12-cyt and MMTV-ADAM12-S transgenic mice exhibited no apparent differences in the mammary gland during development compared with their control littermates (data not shown). Also, neither nulliparous nor multiparous females observed for >18 months developed spontaneous breast tumors. These mice were bred with mice carrying the polyomavirus middle T oncogene (MMTV-PyMT). PyMT expression in the female mammary gland results in rapid development of multifocal mammary adenocarcinomas (20) sharing many features with human breast cancer (21). Using Western blotting, PyMT tumors were found to express little endogenous ADAM12, whereas large amounts of ADAM12 was detected in PyMT breast tumor tissue in which the ADAM12 gene was introduced (Fig. 2B). In female offspring, tumors in ADAM12-cyt/PyMT mice emerged by day 48 (±10), 10 days earlier than PyMT mice (day 58 ± 12; P = 0.0823; Fig. 2C), whereas ADAM12-S/PyMT mice had palpable tumors by day 41 (±5), 9 days earlier than PyMT littermates (day 50 ± 7; P = 0.0003; Fig. 2D). All mice were euthanized at 12 weeks of age, at which time all had developed palpable tumors. The average total tumor mass in ADAM12-cyt/PyMT mice was 2.09 ± 0.62 g compared with 1.22 ± 0.67 g in PyMT littermates (P = 0.0153; Fig. 2E). The average total tumor mass in ADAM12-S/PyMT mice was 2.06 ± 0.97 g compared with 1.15 ± 1.06 g in PyMT littermates (P = 0.0005; Fig. 2F). Thus, enhanced ADAM12 expression in PyMT tumors accelerates tumor development and increases tumor burden.
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cyt– and ADAM12-S–expressing PyMT tumors were compared with PyMT tumors to estimate the relative areas characterized as premalignant or malignant lesions. ADAM12-expressing tumors had significantly larger areas of malignant tumor tissue than did PyMT littermates (P < 0.004) and a reduction in premalignant areas (Fig. 2I and J). The frequency of lung metastasis was also evaluated. In ADAM12-cyt/PyMT mice (n = 12), 77.8% showed lung metastases compared with 25% in their PyMT littermates (n = 12; P < 0.03). In ADAM12-S/PyMT mice, the frequency of metastasis was not significantly different from the PyMT littermates (not shown). These results support the notion that ADAM12 confers increased malignancy in this mouse model.
We next evaluated the degree of cell proliferation and apoptosis in PyMT breast tumors. For 5-bromo-2'-deoxyuridine (BrdUrd) incorporation into tumor cells, no difference was observed between ADAM12-expressing PyMT mice and their littermate controls (11.6 ± 1.8% versus 11.7 ± 1.3% labeled cells, P = 0.42), suggesting the overall rate of tumor cell proliferation was not significantly influenced by ADAM12 expression (Fig. 3A and B). Because the effect of ADAM12 on tumor progression might not be primarily caused by changes in tumor cell proliferation, we asked whether ADAM12 might influence cell survival by examining the level of apoptosis in both tumor and stromal cells. First, we estimated the percent of apoptotic tumor cells using the ApoTag assay (Fig 3C); 1.59 ± 0.29% of tumor cells in ADAM12-cyt/PyMT mice and 4.54 ± 1.01% of tumor cells in PyMT mice were found to be apoptotic (P = 0.0015), suggesting the overall level of tumor cell apoptosis was reduced in ADAM12-expressing PyMT breast tumors. These results correlated with Western blot data demonstrating a reduced level of proapoptotic Bax protein expression in ADAM12-cyt/PyMT tumor tissue compared with tumor tissue of PyMT littermates (Fig. 3D). Next, we examined the influence of ADAM12 on stromal cell apoptosis. Interestingly, we found an increase in apoptotic cells in the stroma of ADAM12-expressing tumors compared with their littermate controls as assessed by hematoxylin staining (data not shown), by ApoTag staining (Fig. 3E-G), and by immunostaining with antibodies to caspase 3 (Fig. 3H). A statistically significant difference in the number of apoptotic stromal cells was observed in ADAM12-cyt/PyMT mice compared with that of PyMT littermates (P = 0.003; Fig. 3I) and in ADAM12-S/PyMT tumors compared with that of PyMT littermates (P = 0.049; Fig. 3J). These results strongly suggest that ADAM12 may accelerate tumor progression by reducing tumor cell apoptosis and by increasing stromal cell apoptosis.
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cyt) or secreted ADAM12-S. Cell proliferation assays showed that ADAM12 decreased growth of nonmalignant cells but had no effect on tumor cell growth (Supplementary Fig. S1). Interestingly, nonmalignant cells of both mesenchymal and epithelial origin (3T3-L1 and CHO-K1, respectively) expressing ADAM12-cyt were more sensitive to apoptosis induced by treatment with TNF or UV-irradiation than their control cells not overexpressing ADAM12 (Fig. 4A and B). As a specificity control, cells were treated with the broad caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-CH2F (zVad-fmk), which completely abolished apoptosis (Fig. 4B). We next tested the effect of adding purified human full-length ADAM12-S on TNF-induced apoptosis. Nonmalignant cells treated with ADAM12-S exhibited an increased sensitivity to apoptosis compared with that of cells not exposed to ADAM12-S (Fig. 4C and D). In sharp contrast, the addition of ADAM12-S to malignant cells (HeLa, A-431, and MG-63) rendered them resistant to TNF-induced apoptosis as assessed by FACS analysis of annexin V– and propidium iodide–positive cells and by quantifying the percentage of condensed nuclei (Fig. 4E and F). These results show that ADAM12 had opposing effects on nonmalignant and malignant tumor cells; nonmalignant cells became more sensitive to apoptosis, whereas malignant cells became more resistant.
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Our findings provide novel insight into the role of ADAM12 in malignant tumors. ADAM12 increased tumor aggressiveness (by decreasing time for tumor onset, increasing tumor burden and metastasis, and increasing the degree of malignancy histologically) and, importantly, decreased tumor cell apoptosis. Unexpectedly, we observed that ADAM12 increased stromal cell apoptosis both in vivo and in vitro. We suggest that increased stromal cell apoptosis might work in concert with increased tumor cell resistance to apoptosis, both favoring tumor progression. More specifically, we propose that increased stromal cell apoptosis contributes to the ongoing remodeling of the stromal compartment. It has been recently suggested that stromal cell apoptosis in human breast carcinomas might be a new biomarker of malignancy (26). In conclusion, we show that ADAM12 facilitates breast cancer progression and we propose that the mechanism involved relates to the ability of ADAM12 to influence the apoptotic potential of tumor and stromal cells differentially.
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Acknowledgments |
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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 Dr. Teijo Kuopio (Jyväskylä Central Hospital, Jyväskylä, Finland) for help with tissue samples; Xiufeng Xu and Eva Engvall for help with generating the first ADAM12 transgenic mice; Brit Valentin, Jacqueline Tybjerg, and Signe Breum for technical assistance; and Linda Raab and Dana Beitner-Johnson for editing the manuscript.
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Footnotes |
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M. Kveiborg, C. Fröhlich, and R. Albrechtsen contributed equally to this work.
Received 1/26/05. Revised 3/ 1/05. Accepted 3/30/05.
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(ADAM12)-deficient mice: involvement of Meltrin in adipogenesis and myogenesis. Mol Cell Biol 2003;23:55–61.) provokes myogenesis in vivo. J Biol Chem 1998;273:157–66.) supports tumor cell adhesion. Am J Pathol 1999;154:1489–501.
. J Biol Chem 2004;279:51601–11.This article has been cited by other articles:
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K. Bourd-Boittin, H. Le Pabic, D. Bonnier, A. L'Helgoualc'h, and N. Theret RACK1, a New ADAM12 Interacting Protein: CONTRIBUTION TO LIVER FIBROGENESIS J. Biol. Chem., September 19, 2008; 283(38): 26000 - 26009. [Abstract] [Full Text] [PDF]
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 N. Kawaguchi, K. Horiuchi, J. D. Becherer, Y. Toyama, P. Besmer, and C. P. Blobel Different ADAMs have distinct influences on Kit ligand processing: phorbol-ester-stimulated ectodomain shedding of Kitl1 by ADAM17 is reduced by ADAM19 J. Cell Sci., March 15, 2007; 120(6): 943 - 952. [Abstract] [Full Text] [PDF]
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